U.S. patent application number 14/916943 was filed with the patent office on 2016-07-07 for antibody which specifically reacts with human integrin a6b4.
This patent application is currently assigned to UNIVERSITY OF MIYAZAKI. The applicant listed for this patent is FUJITA HEALTH UNIVERSITY, PERSEUS PROTEOMICS INC., UNIVERSITY OF MIYAZAKI. Invention is credited to Kazuko KANEDA, Katsushi KOUDA, Gene KUROSAWA, Yoshikazu KUROSAWA, Katsuyuki MITOMO, Kazuhiro MORISHITA, Yoshinori UKAI.
Application Number | 20160194400 14/916943 |
Document ID | / |
Family ID | 52628513 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160194400 |
Kind Code |
A1 |
MORISHITA; Kazuhiro ; et
al. |
July 7, 2016 |
ANTIBODY WHICH SPECIFICALLY REACTS WITH HUMAN INTEGRIN A6B4
Abstract
It is an object of the present invention to provide an
anti-ITGA6/B4 human antibody, which specifically recognizes ITGA6B4
complex expressed on a cell membrane and inhibits the adhesion of
the ITGA6B4 complex to laminin, so as to inhibit adhesion of cancer
cells to a bone marrow niche, and which is also capable of
remarkably enhancing the effects of an anticancer agent on an
anticancer agent resistant strain. The present invention provides
an antibody against integrin A6B4, wherein the antibody
specifically recognizes a human integrin A6B4 complex and inhibits
intercellular adhesion.
Inventors: |
MORISHITA; Kazuhiro;
(Miyazaki-shi, JP) ; KANEDA; Kazuko;
(Miyazaki-shi, JP) ; KUROSAWA; Yoshikazu;
(Toyoake-shi, JP) ; KUROSAWA; Gene; (Tokyo,
JP) ; MITOMO; Katsuyuki; (Tokyo, JP) ; KOUDA;
Katsushi; (Tokyo, JP) ; UKAI; Yoshinori;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MIYAZAKI
FUJITA HEALTH UNIVERSITY
PERSEUS PROTEOMICS INC. |
Miyazaki
Aichi
Tokyo |
|
JP
JP
JP |
|
|
Assignee: |
UNIVERSITY OF MIYAZAKI
Miyazaki-shi, Miyazaki
JP
FUJITA HEALTH UNIVERSITY
Toyoake-shi, Aichi
JP
PERSEUS PROTEOMICS INC.
Tokyo
JP
|
Family ID: |
52628513 |
Appl. No.: |
14/916943 |
Filed: |
September 5, 2014 |
PCT Filed: |
September 5, 2014 |
PCT NO: |
PCT/JP2014/073507 |
371 Date: |
March 4, 2016 |
Current U.S.
Class: |
424/158.1 ;
435/252.3; 435/254.2; 435/320.1; 435/336; 435/419; 435/69.6;
530/387.3; 530/389.2; 536/23.53 |
Current CPC
Class: |
C07K 2317/626 20130101;
A61P 1/00 20180101; C07K 2317/622 20130101; A61P 13/10 20180101;
A61K 31/704 20130101; A61P 11/00 20180101; A61P 15/00 20180101;
C07K 2317/24 20130101; A61K 39/3955 20130101; A61K 39/3955
20130101; A61K 31/365 20130101; C07K 2317/565 20130101; C07K
2317/624 20130101; A61K 31/7068 20130101; A61K 2039/505 20130101;
C07K 2317/73 20130101; C07K 16/2839 20130101; C07K 2317/92
20130101; A61P 17/00 20180101; A61P 35/02 20180101; C07K 2317/76
20130101; C07K 2317/90 20130101; A61P 1/18 20180101; A61P 1/16
20180101; C07K 2317/94 20130101; C07K 2317/21 20130101; C07K
2317/54 20130101; A61P 13/08 20180101; C12N 2510/02 20130101; C07K
2317/55 20130101; A61P 35/00 20180101; A61K 2300/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 31/365 20060101 A61K031/365; A61K 31/704 20060101
A61K031/704; A61K 39/395 20060101 A61K039/395; A61K 31/7068
20060101 A61K031/7068 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 2013 |
JP |
2013-183703 |
Claims
1. An antibody against integrin A6B4, wherein the antibody
specifically recognizes a human integrin A6B4 complex and inhibits
intercellular adhesion.
2. An antibody against integrin A6B4, wherein the antibody
specifically recognizes a human integrin A6B4 complex and increases
a sensitivity of a cancer cell expressing a target antigen to an
anticancer agent.
3. The antibody according to claim 1, which does not substantially
have an influence on the growth of the cell.
4. An antibody against integrin A6B4, wherein the antibody
specifically recognizes a human integrin A6B4 complex and has at
least two or more of the following (a) to (c): (a) an ability to
inhibit intercellular adhesion mediated by integrin A6B4; (b) an
ability to increase a sensitivity of a cancer cell to an anticancer
agent; and (c) a property of substantially not having an influence
on growth of cell.
5. The antibody according to claim 2, wherein the anticancer agent
is selected from the following (i) to (v): (i) an alkylating agent
selected from among ifosfamide, nimustine hydrochloride,
procarbazine hydrochloride, cyclophosphamide, dacarbazine and
thiotepa; (ii) a metabolic antagonist selected from among
cytarabine, enocitabine, gemcitabine hydrochloride, carmofur,
tegafur and fluorouracil; (iii) a plant alkaloid selected from
among etoposide, irinotecan, docetaxel, paclitaxel, vincristine,
vindesine and vinblastine; (iv) an anticancer antibiotic selected
from among actinomycin D, idarubicin, doxorubicin, hydrarubicin,
mitoxantrone and mitomycin C; and (v) a platinum-based formulation
selected from among carboplatin, cisplatin, nedaplatin and
oxaliplatin.
6. The antibody according to claim 1, which is a human antibody or
a humanized antibody.
7. The antibody according to claim 1, which comprises the amino
acid sequence shown in any one of SEQ ID NOS: 1 to 3, 7 to 9, 13 to
15, 19 to 21 and 25 to 27, or an amino acid sequence substantially
identical thereto, as each of a heavy chain first
complementarity-determining region (VH CDR1), a heavy chain second
complementarity-determining region (VH CDR2), or a heavy chain
third complementarity-determining region (VH CDR3).
8. The antibody according to claim 1, which comprises the amino
acid sequence shown in any one of SEQ ID NOS: 1 to 3, 7 to 9, 13 to
15, 19 to 21 and 25 to 27, or an amino acid sequence substantially
identical thereto, as each of a heavy chain first
complementarity-determining region (VH CDR1), a heavy chain second
complementarity-determining region (VH CDR2), or a heavy chain
third complementarity-determining region (VH CDR3), and which also
comprises the amino acid sequence shown in any one of SEQ ID NOS: 4
to 6, 10 to 12, 16 to 18, 22 to 24 and 28 to 30, or an amino acid
sequence substantially identical thereto, as each of a light chain
first complementarity-determining region (VL CDR1), a light chain
second complementarity-determining region (VL CDR2), and a light
chain third complementarity-determining region (VL CDR3).
9. The antibody according to claim 1, which is selected from the
following (i) to (v): (i) an antibody comprising a heavy chain
first complementarity-determining region (VH CDR1) consisting of
the amino acid sequence shown in SEQ ID NO: 1, a heavy chain second
complementarity-determining region (VH CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 2, a heavy chain third
complementarity-determining region (VH CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 3, a light chain first
complementarity-determining region (VL CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 4, a light chain second
complementarity-determining region (VL CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 5, and a light chain third
complementarity-determining region (VL CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 6; (ii) an antibody
comprising a heavy chain first complementarity-determining region
(VH CDR1) consisting of the amino acid sequence shown in SEQ ID NO:
7, a heavy chain second complementarity-determining region (VH
CDR2) consisting of the amino acid sequence shown in SEQ ID NO: 8,
a heavy chain third complementarity-determining region (VH CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 9, a
light chain first complementarity-determining region (VL CDR1)
consisting of the amino acid sequence shown in SEQ ID NO: 10, a
light chain second complementarity-determining region (VL CDR2)
consisting of the amino acid sequence shown in SEQ ID NO: 11, and a
light chain third complementarity-determining region (VL CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 12; (iii)
an antibody comprising a heavy chain first
complementarity-determining region (VH CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 13, a heavy chain second
complementarity-determining region (VH CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 14, a heavy chain third
complementarity-determining region (VH CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 15, a light chain first
complementarity-determining region (VL CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 16, a light chain second
complementarity-determining region (VL CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 17, and a light chain third
complementarity-determining region (VL CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 18; (iv) an antibody
comprising a heavy chain first complementarity-determining region
(VH CDR1) consisting of the amino acid sequence shown in SEQ ID NO:
19, a heavy chain second complementarity-determining region (VH
CDR2) consisting of the amino acid sequence shown in SEQ ID NO: 20,
a heavy chain third complementarity-determining region (VH CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 21, a
light chain first complementarity-determining region (VL CDR1)
consisting of the amino acid sequence shown in SEQ ID NO: 22, a
light chain second complementarity-determining region (VL CDR2)
consisting of the amino acid sequence shown in SEQ ID NO: 23, and a
light chain third complementarity-determining region (VL CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 24; and
(v) an antibody comprising a heavy chain first
complementarity-determining region (VH CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 25, a heavy chain second
complementarity-determining region (VH CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 26, a heavy chain third
complementarity-determining region (VH CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 27, a light chain first
complementarity-determining region (VL CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 28, a light chain second
complementarity-determining region (VL CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 29, and a light chain third
complementarity-determining region (VL CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 30.
10. The antibody according to claim 7, which comprises a deletion,
addition, substitution and/or insertion of one or several amino
acids with respect to the amino acid sequence shown in any one of
SEQ ID NOS: 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 4 to 6,
10 to 12, 16 to 18, 22 to 24 and 28 to 30, and which has an
activity of specifically reacting with human ITGA6B4.
11. The antibody according to claim 1, which is an antibody
fragment selected from the group consisting of Fab, Fab', F (ab')
2, a single chain antibody (scFv), a dimerized V region (Diabody),
a disulfide stabilized V region (dsFv), and a peptide comprising
CDR.
12. DNA encoding the antibody according to claim 7.
13. A recombinant vector comprising the DNA according to claim
12.
14. A transformed cell line obtained by introducing the recombinant
vector according to claim 12 into a host cell.
15. A method for producing an antibody against integrin A6B4,
wherein the antibody specifically recognizes a human integrin A6B4
complex and inhibits intercellular adhesion, which comprises
culturing the transformed cell line according to claim 14 in a
medium, allowing the antibody to generate and accumulate in a
culture, and collecting the antibody from the culture.
16. An inhibitor of cancer cell adhesion, which comprises the
antibody according to claim 1.
17. An inhibitor of cancer cell adhesion, which comprises the
antibody according to claim 1, wherein when the inhibitor is used
in combination with another anticancer agent, it is used to improve
the sensitivity of a cancer cell to the anticancer agent.
18. A pharmaceutical composition comprising the antibody according
to claim 1 and a pharmaceutically acceptable carrier.
19. A pharmaceutical composition comprising the antibody according
to claim 1, wherein in order to increase the drug sensitivity of a
cancer cell and to inhibit the growth of the cancer cell and/or to
kill the cancer cell, a patient who requires such action is
provided with (a) the antibody according to claim 1, and (b) an
anticancer agent selected from among: an alkylating agent selected
from among ifosfamide, nimustine hydrochloride, procarbazine
hydrochloride, cyclophosphamide, dacarbazine and thiotepa; a
metabolic antagonist selected from among cytarabine, enocitabine,
gemcitabine hydrochloride, carmofur, tegafur and fluorouracil; a
plant alkaloid selected from among etoposide, irinotecan,
docetaxel, paclitaxel, vincristine, vindesine and vinblastine; an
anticancer antibiotic selected from among actinomycin D,
idarubicin, doxorubicin, hydrarubicin, mitoxantrone and mitomycin
C; and a platinum-based formulation selected from among
carboplatin, cisplatin, nedaplatin and oxaliplatin, under
conditions and in amounts, which are sufficient for the antibody to
bind to the cancer cell, thereby causing inhibition of the growth
of the cancer cell and/or the death of the cancer cell.
20. The pharmaceutical composition according to claim 19, wherein
the cancer cell has adhered to a bone marrow niche.
21. The pharmaceutical composition according to claim 19, wherein
the cancer is a solid cancer or a blood cancer.
22. The pharmaceutical composition according to claim 21, wherein
the solid cancer is lung cancer, colon cancer, stomach cancer,
bladder cancer, pancreatic cancer, prostate cancer, hepatic cancer,
cervical cancer, uterine cancer, ovarian cancer, breast cancer,
head and neck cancer, or skin cancer.
23. The pharmaceutical composition according to claim 21, wherein
the blood cancer is leukemia, lymphoma, or myeloma.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antibody which
specifically reacts with human integrin A6B4.
BACKGROUND ART
[0002] More than half the adult acute myeloid leukemia (AML)
exhibits resistance to chemotherapy using anticancer agents, and
thus, is refractory. Such refractory leukemia has poor prognosis,
even if born marrow transplantation has been conducted. Thus, it is
urgently required to develop a novel treatment method therefor. To
date, in order to clarify a mechanism of the onset of refractory
leukemia, the present inventors had conducted genomic analysis and
the functional analysis of onset factors thereof, and had isolated
a large number of onset-related factor groups. In particular, the
inventors had conducted the pathological analysis of refractory
leukemia involving an overexpression of the transcriptional factor
EVIL and as a result of comprehensive gene expression analysis,
they had identified an overexpression of .alpha.6-integrin (ITGA6)
and .beta.4 (ITGB4). It had been found that this ITGA6/B4 is highly
expressed not only in leukemia involving an overexpression of EVI1,
but also in a group of patients with leukemia relapse after
completion of the treatment. This phenomenon is generally observed
over the refractory leukemia as a whole. Moreover, it had also been
identified that the overexpression of ITGA6/B4 contributes to the
enhanced adhesion of leukemia cells to osteoblasts and laminin as
an extracellular matrix, and further, to the achievement of
resistance to various types of anticancer agents (Non Patent
Literature 1).
[0003] The enhanced cell adhesion ability of leukemia cells is a
phenomenon that has been well known as Cell Adhesion Mediated Drug
Resistance (CAM-DR), and in recent years, a mechanism whereby
leukemic stem cells (hereinafter referred to as "LSC") take over a
bone marrow niche (hereinafter referred to as "osteoblasts) for
hematopoietic stem cells (hereinafter referred to as "HSC") (which
is adhesion involving utilization of a bone marrow niche
environment), and achieve resistance to anticancer agents, has been
revealed. An integrin family associated with adhesion of HSC to the
bone marrow niche has is mainly VLA-4 (ITGA4/B1) (Non Patent
Literature 2), but it has been found that LSC depends on ITGA6B4.
Accordingly, it is considered that the development of a treatment
method, which targets a specific adhesion molecule ITGA6/B4 that is
associated with CAM-DR leading to incurability, would lead to the
development of a method for effectively treating refractory
leukemia, for which there have been no conventional treatment
methods so far (Patent Literature 1).
[0004] An antibody capable of reacting against ITGA6B4 has been
manufactured by Biogen IDEC MA, Inc., and has been reported in
2007. This antibody has been reported to have an ability to inhibit
the growth of cells and inhibit adhesion of the cells to laminin
(Patent Literature 2).
[0005] The present inventors have searched for an antigen
(antibody) specific to cancer cells by a method for comprehensively
isolating a human antibody, which targets many cancer cells, based
on the Phage Display method (Patent Literature 3). As a result,
ITGA6/B4 (antibody) has been included in the obtained 29 types of
targets, and the validity of ITGA6/B4 as an antibody drug target
has been proved also from another viewpoint (Non Patent Literature
3).
[0006] From the viewpoint of the side effects of a pharmaceutical
product, since chemotherapeutic agents are not specific to cancer
cells, but also exhibit effects on cells with activated cellular
division, including normal cells, it has been conventionally very
important to control administration of the agents. On the other
hand, since molecular-targeted agents have been selectively
designed to specific targeted molecules, these agents are
characterized in that their side effects are reduced in comparison
to the aforementioned chemotherapeutic agents. Antibodies are also
molecular-targeted agents, and thus, when compared with enzyme
inhibitors (kinase inhibitors) and the like, the specificity of
such antibodies to targets has been improved. With such target
specificity, antibody drugs are anticipated to have reduced side
effects, but individual antibody drugs are observed to have
characteristic side effects. It is considered that some side
effects are caused by induction of immune responses such as ADCC
(antibody-dependent cellular cytotoxicity) or CDC
(complement-dependent cytotoxicity), which are derived from the
properties of antibody molecules, and that some other side effects
are caused by agonistic or antagonistic activities induced as a
result of action on target molecules. Moreover, in recent years,
antibody drugs have exhibited strong effects as a result of
reinforced immune responses or drug modification. At the same time,
however, considerable attention should be paid on the side effects
of the antibody drugs.
PRIOR ART LITERATURES
Patent Literatures
[0007] Patent Literature 1: WO2010/098166 [0008] Patent Literature
2: WO2008/127655 [0009] Patent Literature 3: Japanese Patent No.
4870348
Non Patent Literatures
[0009] [0010] Non Patent Literature 1: The Increased Expression of
Integrin .alpha.6 (ITGA6) Enhances Drug Resistance in EVI1.sup.high
Leukemia, Yamakawa, N., et al., ProsOne Vol. 7 Issue 1 e30706
(2012) [0011] Non Patent Literature 2: Interaction between
leukemic-cell VLA-4 and stromal fibronectin is a decisive factor
for minimal residual disease of acute myelogenous leukemia.
Matsunaga, T., et al., Nat Med; 9: 1158-65. (2003) [0012] Non
Patent Literature 3: Comprehensive screening for antigens
overexpressed on carcinomas via isolation of human mAbs that may be
therapeutic, Kurosawa G et al., Proc Nat Acad Sci USA 105,
7287-7292 <2008>
SUMMARY OF INVENTION
Object to be Solved by the Invention
[0013] It is an object of the present invention to provide an
anti-ITGA6B4 human antibody, which specifically recognizes an
integrin A6B4 (ITGA6B4) complex expressed on a cell membrane and
inhibits the adhesion of the ITGA6B4 complex to laminin, so as to
inhibit adhesion of cancer cells to a bone marrow niche, and which
is also capable of remarkably enhancing the effects of an
anticancer agent on an anticancer agent resistant strain. In
addition, it is another object of the present invention to provide
an inhibitor of cancer cell adhesion and a pharmaceutical
composition, each of which comprises the above-described
anti-ITGA6/B4 human antibody with a few side effects upon the use
thereof.
Means for Solving the Object
[0014] The present inventors have conducted intensive studies
directed towards achieving the above-described objects. The
inventors have focused on the fact that refractory leukemic stem
cells adhere to a bone marrow niche so as to obtain resistance to
anticancer agents, and they have assumed that the effects of
anticancer agents can be improved by inhibiting stable adhesion of
the stem cells to the bone marrow niche, thereby releasing the stem
cells or inhibiting the adhesion thereof to the niche (this
phenomenon is referred to as "turn out"). Based on the concept that
antibody molecules would be useful for turning refractory leukemic
stem cells out of a niche, the inventors have searched for an
antibody having an activity of recognizing a human integrin A6B4
complex and inhibiting adhesion of cancer cells to a niche, so as
to increase the sensitivity of the cancer cells to anticancer
agents.
[0015] The present inventors have established a method for
isolating a complete human antibody against an intact protein on
the surface of a cell, which is based on the phage display method.
Since this method uses a phage library retaining an enormous
repertoire, it has a high variety of antibodies. In addition, since
this method can directly screen cancer cells, even an antibody
against a complex protein on the cell surface can be easily
obtained by this method. In the present invention, by applying this
method, a plurality of antibodies against an ITGA6B4 complex
expressed on a cancer cell membrane has been obtained. Thereafter,
antibodies exhibiting cancer specificity, which react with a large
number of cancer cells, including leukemic stem cells, kidney
cancer cells, lung cancer cells, etc., have been selected.
Thereafter, complete human IgG antibodies have been produced from
the obtained antibodies according to a DNA recombination
technique.
[0016] Subsequently, based on the idea that since refractory
leukemic stem cells adhere to a bone marrow niche to obtain
resistance to anticancer agents, the effects of the anticancer
agents can be improved by "turning the stem cells out of" the bone
marrow niche, and that antibodies are useful for such "turning the
stem cells out of" the niche, the present inventors have conceived
of a "turn-out treatment" of using antibodies. In the case of this
treatment, mainly necessary medicinal effect is inhibition of
adhesion of the stem cells to the bone marrow, and there is a fear
that other auxiliary effects would cause unexpected action on all
cells that express ITGA6B4. Hence, in the present invention, an
anti-ITGA6BA antibody, which does not affect the growth of cells,
has been selected. Based on the aforementioned thought, the present
inventors have conducted basic studies regarding the "turn-out
treatment," using a human antibody. As a result, the inventors have
succeeded in confirming, using a model system, that the
anti-ITGA6B4 antibody of the present invention is able to inhibit
adhesion of leukemic stem cells to a bone marrow niche, and that an
antibody having an adhesion-inhibiting action reduces the
resistance of the cancer cells to anticancer agents, thereby
completing the present invention.
[0017] The present invention provides the following invention.
(1) An antibody against integrin A6B4, wherein the antibody
specifically recognizes a human integrin A6B4 complex and inhibits
intercellular adhesion. (2) An antibody against integrin A6B4,
wherein the antibody specifically recognizes a human integrin A6B4
complex and increases a sensitivity of a cancer cell expressing a
target antigen to an anticancer agent. (3) The antibody according
to (1) or (2), which does not substantially have an influence on
the growth of the cell. (4) An antibody against integrin A6B4,
wherein the antibody specifically recognizes a human integrin A6B4
complex and has at least two or more of the following (a) to (c):
(a) an ability to inhibit intercellular adhesion mediated by
integrin A6B4; (b) an ability to increase a sensitivity of a cancer
cell to an anticancer agent; and (c) a property of substantially
not having an influence on growth of cell. (5) The antibody
according to (2) or (4), wherein the anticancer agent is selected
from the following (i) to (v): (i) an alkylating agent selected
from among ifosfamide, nimustine hydrochloride, procarbazine
hydrochloride, cyclophosphamide, dacarbazine and thiotepa; (ii) a
metabolic antagonist selected from among cytarabine, enocitabine,
gemcitabine hydrochloride, carmofur, tegafur and fluorouracil;
(iii) a plant alkaloid selected from among etoposide, irinotecan,
docetaxel, paclitaxel, vincristine, vindesine and vinblastine; (iv)
an anticancer antibiotic selected from among actinomycin D,
idarubicin, doxorubicin, hydrarubicin, mitoxantrone and mitomycin
C; and (v) a platinum-based formulation selected from among
carboplatin, cisplatin, nedaplatin and oxaliplatin. (6) The
antibody according to any one of (1) to (5), which is a human
antibody or a humanized antibody. (7) The antibody according to any
one of (1) to (6), which comprises the amino acid sequence shown in
any one of SEQ ID NOS: 1 to 3, 7 to 9, 13 to 15, 19 to 21 and 25 to
27, or an amino acid sequence substantially identical thereto, as
each of a heavy chain first complementarity-determining region (VH
CDR1), a heavy chain second complementarity-determining region (VH
CDR2), or a heavy chain third complementarity-determining region
(VH CDR3). (8) The antibody according to any one of (1) to (7),
which comprises the amino acid sequence shown in any one of SEQ ID
NOS: 1 to 3, 7 to 9, 13 to 15, 19 to 21 and 25 to 27, or an amino
acid sequence substantially identical thereto, as each of a heavy
chain first complementarity-determining region (VH CDR1), a heavy
chain second complementarity-determining region (VH CDR2), or a
heavy chain third complementarity-determining region (VH CDR3), and
which also comprises the amino acid sequence shown in any one of
SEQ ID NOS: 4 to 6, 10 to 12, 16 to 18, 22 to 24 and 28 to 30, or
an amino acid sequence substantially identical thereto, as each of
a light chain first complementarity-determining region (VL CDR1), a
light chain second complementarity-determining region (VL CDR2),
and a light chain third complementarity-determining region (VL
CDR3). (9) The antibody according to any one of (1) to (8), which
is selected from the following (i) to (v): (i) an antibody
comprising a heavy chain first complementarity-determining region
(VH CDR1) consisting of the amino acid sequence shown in SEQ ID NO:
1, a heavy chain second complementarity-determining region (VH
CDR2) consisting of the amino acid sequence shown in SEQ ID NO: 2,
a heavy chain third complementarity-determining region (VH CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 3, a
light chain first complementarity-determining region (VL CDR1)
consisting of the amino acid sequence shown in SEQ ID NO: 4, a
light chain second complementarity-determining region (VL CDR2)
consisting of the amino acid sequence shown in SEQ ID NO: 5, and a
light chain third complementarity-determining region (VL CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 6; (ii)
an antibody comprising a heavy chain first
complementarity-determining region (VH CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 7, a heavy chain second
complementarity-determining region (VH CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 8, a heavy chain third
complementarity-determining region (VH CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 9, a light chain first
complementarity-determining region (VL CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 10, a light chain second
complementarity-determining region (VL CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 11, and a light chain third
complementarity-determining region (VL CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 12; (iii) an antibody
comprising a heavy chain first complementarity-determining region
(VH CDR1) consisting of the amino acid sequence shown in SEQ ID NO:
13, a heavy chain second complementarity-determining region (VH
CDR2) consisting of the amino acid sequence shown in SEQ ID NO: 14,
a heavy chain third complementarity-determining region (VH CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 15, a
light chain first complementarity-determining region (VL CDR1)
consisting of the amino acid sequence shown in SEQ ID NO: 16, a
light chain second complementarity-determining region (VL CDR2)
consisting of the amino acid sequence shown in SEQ ID NO: 17, and a
light chain third complementarity-determining region (VL CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 18; (iv)
an antibody comprising a heavy chain first
complementarity-determining region (VH CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 19, a heavy chain second
complementarity-determining region (VH CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 20, a heavy chain third
complementarity-determining region (VH CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 21, a light chain first
complementarity-determining region (VL CDR1) consisting of the
amino acid sequence shown in SEQ ID NO: 22, a light chain second
complementarity-determining region (VL CDR2) consisting of the
amino acid sequence shown in SEQ ID NO: 23, and a light chain third
complementarity-determining region (VL CDR3) consisting of the
amino acid sequence shown in SEQ ID NO: 24; and (v) an antibody
comprising a heavy chain first complementarity-determining region
(VH CDR1) consisting of the amino acid sequence shown in SEQ ID NO:
25, a heavy chain second complementarity-determining region (VH
CDR2) consisting of the amino acid sequence shown in SEQ ID NO: 26,
a heavy chain third complementarity-determining region (VH CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 27, a
light chain first complementarity-determining region (VL CDR1)
consisting of the amino acid sequence shown in SEQ ID NO: 28, a
light chain second complementarity-determining region (VL CDR2)
consisting of the amino acid sequence shown in SEQ ID NO: 29, and a
light chain third complementarity-determining region (VL CDR3)
consisting of the amino acid sequence shown in SEQ ID NO: 30. (10)
The antibody according to (7) or (8), which comprises a deletion,
addition, substitution and/or insertion of one or several amino
acids with respect to the amino acid sequence shown in any one of
SEQ ID NOS: 1 to 3, 7 to 9, 13 to 15, 19 to 21, 25 to 27, 4 to 6,
10 to 12, 16 to 18, 22 to 24 and 28 to 30, and which has an
activity of specifically reacting with human ITGA6B4. (11) The
antibody according to any one of (1) to (10), which is an antibody
fragment selected from the group consisting of Fab, Fab', F (ab')
2, a single chain antibody (scFv), a dimerized V region (Diabody),
a disulfide stabilized V region (dsFv), and a peptide comprising
CDR. (12) DNA encoding the antibody according to any one of (7) to
(10). (13) A recombinant vector comprising the DNA according to
(12). (14) A transformed cell line obtained by introducing the
recombinant vector according to (12) into a host cell. (15) A
method for producing the antibody according to any one of (1) to
(11), which comprises culturing the transformed cell line according
to (14) in a medium, allowing the antibody according to any one of
(1) to (11) to generate and accumulate in a culture, and collecting
the culture from the culture. (16) An inhibitor of cancer cell
adhesion, which comprises the antibody according to any one of (1)
to (11). (17) An inhibitor of cancer cell adhesion, which comprises
the antibody according to any one of (1) to (11), wherein when the
inhibitor is used in combination with another anticancer agent, it
is used to improve the sensitivity of a cancer cell to the
anticancer agent. (18) A pharmaceutical composition comprising the
antibody according to any one of (1) to (11) and a pharmaceutically
acceptable carrier. (19) A pharmaceutical composition comprising
the antibody according to any one of (1) to (11), wherein [0018] in
order to increase the drug sensitivity of a cancer cell and to
inhibit the growth of the cancer cell and/or to kill the cancer
cell, a patient who requires such action is provided with [0019]
(a) the antibody according to any one of claims 1 to 11, and [0020]
(b) an anticancer agent selected from among: an alkylating agent
selected from among ifosfamide, nimustine hydrochloride,
procarbazine hydrochloride, cyclophosphamide, dacarbazine and
thiotepa; a metabolic antagonist selected from among cytarabine,
enocitabine, gemcitabine hydrochloride, carmofur, tegafur and
fluorouracil; a plant alkaloid selected from among etoposide,
irinotecan, docetaxel, paclitaxel, vincristine, vindesine and
vinblastine; an anticancer antibiotic selected from among
actinomycin D, idarubicin, doxorubicin, hydrarubicin, mitoxantrone
and mitomycin C; and a platinum-based formulation selected from
among carboplatin, cisplatin, nedaplatin and oxaliplatin, [0021]
under conditions and in amounts, which are sufficient for the
antibody to bind to the cancer cell, thereby causing inhibition of
the growth of the cancer cell and/or the death of the cancer cell.
(20) The pharmaceutical composition according to (19), wherein the
cancer cell has adhered to a bone marrow niche. (21) The
pharmaceutical composition according to (19) or (20), wherein the
cancer is a solid cancer or a blood cancer. (22) The pharmaceutical
composition according to (21), wherein the solid cancer is lung
cancer, colon cancer, stomach cancer, bladder cancer, pancreatic
cancer, prostate cancer, hepatic cancer, cervical cancer, uterine
cancer, ovarian cancer, breast cancer, head and neck cancer, or
skin cancer. (23) The pharmaceutical composition according to (21),
wherein the blood cancer is leukemia, lymphoma, or myeloma.
[0022] According to the present invention, a method for inhibiting
or treating cancer, which comprises administering the
above-described antibody of the present invention to a subject, is
further provided.
[0023] According to the present invention, a method for enhancing
the effects of an anticancer agent, which comprises administering
the above-described antibody of the present invention to a subject,
is further provided.
[0024] According to the present invention, a method for inhibiting
cell adhesion of cancer cells, which comprises administering the
above-described antibody of the present invention to a subject, is
further provided.
[0025] According to the present invention, use of the
above-described antibody of the present invention for production of
an inhibitor of cancer cell adhesion or a pharmaceutical
composition, is further provided.
Effects of Invention
[0026] The antibody of the present invention is able to
specifically recognize a human ITGA6B4 complex and specifically
recognize a refractory cancer cell, which expresses an ITGA6B4
complex on the cell membrane thereof. The antibody of the present
invention is also able to inhibit adhesion of cancer cells to a
bone marrow niche, and enhance the effects of anticancer agents to
anticancer agent resistant strains. Moreover, since the antibody of
the present invention has the property of not affecting the growth
of cells by itself, it is able to reduce unexpected effects,
namely, side effects.
BRIEF DESCRIPTION OF DRAWINGS
[0027] FIG. 1 shows two bands obtained as a result of the
immunoprecipitation performed in Example 5.
[0028] FIG. 2 shows that ITGA6 has been obtained as a result of the
antigen identification with a mass spectrometer performed in
Example 6. The present data consist of the output view obtained
when the mass data obtained using a mass spectrometer (LCQ
manufactured by Thermo) have been analyzed using the analysis
software Mascot search (MATRIX SCIENCE).
[0029] FIG. 3 shows that ITGB4 has been obtained as a result of the
antigen identification with a mass spectrometer performed in
Example 6. The present data consist of the output view obtained
when the mass data obtained using a mass spectrometer (LCQ
manufactured by Thermo) have been analyzed using the analysis
software Mascot search (MATRIX SCIENCE).
[0030] FIG. 4 shows the results obtained by studying the reactivity
of the IgG antibody produced in Example 3 with a purified soluble
human ITGA6B4 antigen (Recombinant Human Integrin alpha 6
(.times.1) beta 4, R & D) according to ELISA.
[0031] FIG. 5 is a view showing that overexpression of
.alpha.6-integrin (ITGA6) and .beta.4 (ITGB4) has been identified
by the comprehensive gene expression analysis of refractory
leukemia.
[0032] FIG. 6 is a view showing that adhesion of leukemia cells via
ITGA6/B4 is necessary for the leukemia cells to obtain resistance
to anticancer agents. The figure shows that the anti-ITGA6/B4
antibody of the present invention contributes to attenuation of the
adhesion ability.
[0033] FIG. 7 shows the results obtained by performing a cell
adhesion inhibition test on four AML cell lines, namely, CSD/AML1,
Kasumi3, MOLM1, and HNT34. The figure shows that the adhesiveness
of the cells is reduced by administration of an anti-ITGA6/B4
antibody.
[0034] FIG. 8 shows influence caused by the combined use of an
anti-ITGA6/B4 antibody with anticancer agents [AraC (cytarabine),
DXR (doxorubicin), and VP16 (etoposide)]. It is found that the
anti-ITGA6/B4 antibody enhances the effects of the anticancer
agents.
[0035] FIG. 9 shows the results obtained by examining the
expression level of ITGA6B4 in leukemic stem cells.
[0036] FIG. 10 shows the results obtained by evaluating a change in
the reactivity of antibodies in a gene expression inhibition test,
where cells, into which ITGA6 and ITGB4 genes had been introduced
by shRNA, were used.
[0037] FIG. 11 shows the results obtained by examining the
infiltration-inhibiting effect by the combined use of an antibody
and an anticancer agent, using vein transplant models. It has been
demonstrated that the infiltration of leukemia cells into the bone
marrow is significantly inhibited by administration of an
anti-ITGA6BA antibody in both cases of a single administration and
a combined use.
[0038] FIG. 12-1 shows the results obtained by examining the effect
of an ITGA6B4 antibody to inhibit the in vitro growth of cells. In
addition, in FIG. 12-2, the results are shown based on a cell
growth level, in which PBS is defined as 100, and it has been
demonstrated that the anti-ITGA6BA antibody does not have the
effect of inhibiting cell growth when it is used alone.
[0039] FIG. 13 shows the in vivo antitumor effects by using vein
transplant models. The extension of the survival rate was observed
by the combined use of the antibody and the anticancer agent.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0040] Hereinafter, the present invention will be described more in
detail. It is to be noted that, in the present description, there
is a case where an integrin .alpha.6.beta.4 complex that is an
antigen specifically recognized by the antibody of the present
invention may also be referred to as "ITGA6/B4," "ITGA6B4,"
"ITGA6B4 complex," "A6B4 complex," or the like.
[0041] The present inventors have conducted the pathological
analysis of refractory leukemia, and as a result of a comprehensive
gene expression analysis, the inventors have identified
overexpression of .alpha.6-integrin (ITGA6) and .beta.4 (ITGB4).
Moreover, it has been found that this ITGA6/B4 is overexpressed,
not only in EVI1 overexpression leukemia, but also in a group of
patients with leukemia relapse after completion of the treatment
(FIG. 5). Since this is a phenomenon generally observed in the
refractory leukemia as a whole. It can be anticipated that this
phenomenon will be particularly effective for the treatment of
leukemia. According to the antibody of the present invention, the
drug sensitivity of cancer showing resistance to the existing
anticancer agents can be increased by inhibiting adhesion of the
cancer cells to a bone marrow niche via ITGA6B4, and as a result,
the effects of the anticancer agents can be restored or increased,
and it becomes possible to carry out an effective treatment. With
regard to the antibody of the present invention, an antibody, which
particularly has high specificity and high affinity for the cancer
cells of a patient and retains the effect of enhancing the
aforementioned sensitivity to anticancer agents, is selected. Thus,
high therapeutic effects on cancer cells can be obtained. The
antibody of the present invention is characterized in that it is an
antibody against an ITGA6B4 complex, in that it is able to carry
out, in particular, the above-described "turn-out treatment", and
in that it is able to significantly enhance the sensitivity of
cancer cells to the existing anticancer agents. Although Patent
Literature 1 describes the effects of integrin A6 or integrin B4 as
a single use, the antibody of the present invention is an antibody
against an integrin A6B4 complex. Since the antibody of the present
invention recognizes integrin A6B4 and is modified to be an
antibody suitable for the "turn-out treatment" proposed by the
present invention, it can be anticipated that the present antibody
will have significant therapeutic effects also on patients with
leukemia relapse.
[0042] Unless otherwise specified in the present description,
scientific terms used regarding the present invention have meanings
that are generally understood by a person skilled in the art. In
general, nomenclatures and techniques applied to the cell and
tissue culture, molecular biology, immunology, microbiology,
genetics, protein and nucleic acid chemistry, and hybridization,
which are described in the present description, are well known in
the present technical field, and thus, are commonly used.
[0043] The methods and techniques of the present invention are
carried out in accordance with conventional methods that are well
known in the present technical field, in such ways as described in
a variety of general reference documents cited and discussed
throughout the present description and more specific reference
documents, unless otherwise specified.
Integrin A6B4 (ITGA6B4)
[0044] It has been known that integrin A6B4 (ITGA6B4) is a complex
consisting of integrin .alpha.6 and integrin .beta.4, and that the
ITGA6B4 complex is associated with a large number of phenomena,
such as interaction with an extracellular matrix, intercellular
adhesion, adhesion of cells to tissues, and signaling. It has been
known that integrin .alpha.6 forms a complex, not only with
.beta.4, but also with .beta.1, so as to construct a complex
referred to as "VLA6." The present antigen has been known as CD49f,
and has been considered to be present in platelets or megakaryocyte
and to be weakly expressed in a subpopulation of T cells. On the
other hand, the complex as a target of the present study has been
considered to be mainly expressed in normal cells of the bronchial
epithelium, and also in the cancer cells of many cancers, including
colon cancer as a typical example.
[0045] ITGA6B4 is not particularly limited in terms of structure,
and thus, it totally means human ITGA6B4 including a monomer, a
polymer, an intact form expressed on a cell membrane, a soluble
form constituted in an extracellular region, a truncated form, a
mutation form caused by genetic mutation, deletion, etc., and a
form that has undergone posttranslational modification by
phosphorylation or the like.
React and Reactivity
[0046] The terms "to react" and "reactivity" have the same meanings
in the present description, unless otherwise specified. That is,
these terms mean that an antibody recognizes an antigen. The
antigen used herein may be any of an intact ITGA6B4 expressed on a
cell membrane, a truncated form, and a soluble form. In addition,
the antigen may be either an ITGA6B4 having a three-dimensional
structure or a denatured ITGA6B4. Examples of a means for examining
reactivity include flow cytometry (FACS), enzyme-linked
immunosorbent assay (ELISA), Western blotting, microfluorescence
measuring technique (FMAT), surface plasmon resonance (BIAcore),
immunostaining, and immunoprecipitation.
[0047] The antibody used in flow cytometry may be either an
antibody labeled with a fluorescent substance such as FITC or with
biotin, or an unlabeled antibody. A fluorescently-labeled avidin, a
fluorescently-labeled anti-human immunoglobulin antibody, or the
like is used, depending on the presence or absence of labeling of
the antibody used and the type thereof. Reactivity can be evaluated
by adding a sufficient amount of anti-ITGA6B4 antibody (generally
having a final concentration of 0.01 to 10 .mu.g/mL) to an analyte,
and then by comparing the obtained reactivity with the reactivity
with a negative control antibody or a positive control
antibody.
Antibody
[0048] In the present description, the following abbreviations (in
the parentheses) are used in accordance with the customs, as
necessary:
Heavy chain (H chain), light chain (L chain), heavy chain variable
region (VH), light chain variable region (VL), and
complementarity-determining region (CDR).
[0049] In the present description, the term "antibody" has the same
definitions as immunoglobulin, and should be understood as
generally known in the present technical field. Specifically, the
term "antibody" is not limited by any given specific method for
producing the antibody. For example, the term "antibody" includes,
but is not limited to, a recombinant antibody, a monoclonal
antibody, and a polyclonal antibody.
[0050] In the present description, the term "human antibody is used
to mean any given antibody, in which the sequences of a variable
region and a constant region are human sequences. This term
includes antibodies which have human sequences and are modified,
for example, to remove cysteine that may cause a possible decrease
in immunogenicity, an increase in affinity, and undesirable
folding. This term also includes antibodies produced in non-human
cells by recombination, which enable glycosylation that is not
specific to human cells. These antibodies can be prepared in
various ways.
[0051] In the present description, the term "humanized antibody"
means a non-human-derived antibody, in which amino acid residues
characteristic for a non-human antibody sequence are substituted
with residues found in corresponding positions of a human antibody.
This "humanization" process is considered to reduce the
immunogenicity of the obtained antibody in human. It would be
understood that a non-human-derived antibody can be humanized using
a technique well known in the present technical field. Please refer
to, for example, Winter et al., Immunol. Today 14: 43-46 (1993).
The target antibody can be produced by an engineering approach via
a recombination DNA technique of substituting CH1, CH2, CH3, a
hinge domain, and/or a framework domain with those of the
corresponding human sequence. For example, WO92/02190, and U.S.
Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350 and
5,777,085 can be referred. In the present description, the term
"humanized antibody" includes a chimeric human antibody and a
CDR-grafted antibody, within the definitions thereof.
[0052] The sequence of a framework region (FR) in a variable region
of the antibody of the present invention is not particularly
limited, unless it substantially affects the specific binding
ability of the antibody to the corresponding antigen. The FR region
of a human antibody is preferably used, but it is also possible to
use FR regions of animal species other than humans (e.g. a mouse, a
rat, etc.).
[0053] In the present description, the term "phage antibody" is
used to mean a scFv antibody generated from phage. That is, the
phage antibody is an antibody fragment comprising the amino acid
sequence of VH and VL. This fragment may comprise an amino acid
sequence serving as a tag, as well as amino acids serving as a
linker.
[0054] In one aspect, the antibody of the present invention
comprises a constant region as well as a variable region (e.g. IgG
antibody). The sequence of such a constant region is not
particularly limited. For example, the constant region of a known
human antibody can be used. The heavy chain constant region (CH) of
a human antibody is not particularly limited, as long as it belongs
to a human immunoglobulin (hereinafter referred to as "hIg"). Those
of hIgG class are preferable, and any one of subclasses belonging
to hIgG class, such as hIgG1, hIgG2, hIgG3 or hIgG4, may be used.
On the other hand, the light chain constant region (CL) of a human
antibody is not particularly limited, as long as it belongs to hIg,
and those of .kappa. class or .lamda. class can be used. In
addition, constant regions of animal species other than humans
(e.g. a mouse or a rat) can also be used.
[0055] As the amino acid sequence of the variable region (CDR
sequence and/or FR sequence) or constant region of the antibody of
the present invention, the amino acid sequence of the original FR
or constant region, from which the present antibody is derived, may
be directly used. Otherwise, an amino acid sequence comprising a
deletion, addition, substitution and/or insertion of one or several
amino acids (for example, 1 to 8, preferably 1 to 5, more
preferably 1 to 3, and particularly preferably one or two amino
acids) may also be used, as long as the resulting antibody of the
present invention can specifically recognize human ITGA6B4.
[0056] The antibody of the present invention preferably comprises
the amino acid sequence shown in any one of SEQ ID NOS: 1 to 3, 7
to 9, 13 to 15, 19 to 21 and 25 to 27, as each of a heavy chain
first complementarity-determining region (VH CDR1), a heavy chain
second complementarity-determining region (VH CDR2), and a heavy
chain third complementarity-determining region (VH CDR3), and it
also comprises the amino acid sequence shown in any one of SEQ ID
NOS: 4 to 6, 10 to 12, 16 to 18, 22 to 24 and 28 to 30, as each of
a light chain first complementarity-determining region (VL CDR1), a
light chain second complementarity-determining region (VL CDR2),
and a light chain third complementarity-determining region (VL
CDR3).
[0057] Moreover, the antibody of the present invention may also
comprise an amino acid sequence that is substantially identical to
the amino acid sequence shown in any one of SEQ ID NOS: 1 to 3, 7
to 9, 13 to 15, 19 to 21 and 25 to 27, as each of a heavy chain
first complementarity-determining region (VH CDR1), a heavy chain
second complementarity-determining region (VH CDR2), and a heavy
chain third complementarity-determining region (VH CDR3), and it
may further comprise an amino acid sequence that is substantially
identical to the amino acid sequence shown in any one of SEQ ID
NOS: 4 to 6, 10 to 12, 16 to 18, 22 to 24 and 28 to 30, as each of
a light chain first complementarity-determining region (VL CDR1), a
light chain second complementarity-determining region (VL CDR2),
and a light chain third complementarity-determining region (VL
CDR3).
[0058] The phrase "can specifically recognize human ITGA6B4" is
used herein to mean that the antibody of the present invention has
an activity of specifically reacting with human integrin A6B4, as
with the antibody before modification. This antigen may be either
intact ITGA6B4 expressed on a cell membrane, or a truncated form or
a soluble form. Furthermore, the antigen may be either ITGA6B4
retaining a three-dimensional structure, or a denatured ITGA6B4.
Examples of a means for examining binding activity include flow
cytometry (FACS), enzyme-linked immunoadsorbent assay (ELISA),
Western-blot, fluorescence microassay technology (FMAT), and
surface plasmon resonance (BIAcore).
[0059] In the present invention, the phrase "as with the antibody
before modification" does not necessarily mean the same level of
activity. The activity may be increased, or the activity may also
be decreased, as long as the antibody has the activity. An antibody
having a decreased activity may be an antibody having an activity
that is, for example, 30% or more, preferably 50% or more, more
preferably 80% or more, further preferably 90% or more, and
particularly preferably 95% or more of the activity of the original
antibody.
[0060] The antibody of the present invention may comprise a
substitution, deletion, addition and/or insertion of one or several
amino acids with respect to the amino acid sequence of a variable
region (a CDR sequence and/or an FR sequence), as long as it has an
activity of specifically reacting with human integrin A6B4. As a
method for preparing the amino acid sequence of an antibody
specifically reacting with human integrin A6B4, which comprises a
deletion, addition, substitution and/or insertion of one or several
amino acids (for example, 1 to 8, preferably 1 to 5, more
preferably 1 to 3, and particularly preferably 1 or 2 amino acids),
a method of introducing a mutation into a protein has been well
known to a person skilled in the art. For instance, such a skilled
person could prepare a mutant antibody functionally equivalent to
an antibody specifically reacting with human integrin A6B4 by
appropriately introducing a mutation into the amino acid sequence
of the aforementioned antibody according to a site-directed
mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, an DNA
kagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber
method for site-directed mutagenesis. Gene 152, 271-275, Zoller, M
J, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500,
Kramer, W, Drutsa, V, Jansen, H W, Kramer, B, Pflugfelder, M, and
Fritz, H J (1984) The gapped duplex DNA approach to
oligonucleotide-directed mutation construction. Nucleic Acids Res.
12, 9441-9456, Kramer W, and Fritz HJ (1987)
Oligonucleotide-directed construction of mutations via gapped
duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Rapid
and efficient site-specific mutagenesis without phenotypic
selection. Proc NAML Acad Sci USA. 82, 488-492), etc.
[0061] As such, an antibody, which comprises a mutation of one or
several amino acids in a variable region thereof and specifically
reacts with human integrin A6B4, is also included in the antibody
of the present invention.
[0062] The antibody of the present invention is not limited by its
origin, and it may be an antibody derived from any animal, such as
a human antibody, a mouse antibody, or a rat antibody. Also, the
present antibody may be a chimeric antibody or a humanized
antibody.
[0063] The antibodies of the present invention may be different
from one another in terms of amino acid sequence, molecular weight,
isoelectric point, the presence or absence of a sugar chain or the
form thereof, etc., depending on the after-mentioned cells or hosts
that produce the antibodies, or a purification method. As long as
the obtained antibody has an activity equivalent to the antibody of
the present invention, it is included in the present invention. For
example, an antibody that undergoes a modification after it has
been translated to the amino acid sequence described in the present
description is also included in the present invention. Moreover, an
antibody that has undergone a posttranslational modification on a
site other than those for the known posttranslational modification
is also included in the present invention, as long as it has an
activity equivalent to the antibody of the present invention.
Furthermore, when the antibody of the present invention is allowed
to express in prokaryotic cells such as Escherichia coli, a
methionine residue is added to the N-terminus of the amino acid
sequence of the original antibody. The antibody of the present
invention includes such an antibody as well. An antibody that has
undergone a posttranslational modification on a site other than
those for the known posttranslational modification is also included
in the present invention, as long as it has an activity equivalent
to the antibody of the present invention.
Production of Antibody
[0064] (1) Production of scFv Reacting with Antigen Using Phage
Display Library
[0065] The antibody of the present invention can be prepared by
several methods known in the present technical field. For example,
using a phage display technique, a library comprising a repertoire
of antibodies having various affinity for ITGA6B4 can be provided.
Subsequently, such a library can be screened to identify and
isolate antibodies against ITGA6B4. Preferably, the phage library
is a scFv phage display library that is generated using human VL
and VH cDNA that has been prepared from mRNA isolated from human B
cells. A method of preparing and screening such a library is known
in the present technical field. A genetic substance is recovered
from phage clones exhibiting reactivity that have been screened
using a human ITGA6B4 as an antigen. By analyzing the selected
phage gene, the DNA sequences of VH and VL encoding the variable
region of a human antibody binding to the antigen can be
determined. Using this scFv sequence, IgG is prepared from scFv, so
as to obtain a human antibody.
(2) Preparation of IgG from scFv (Preparation of Human
Antibody)
[0066] An H chain or L chain expression vector is produced, and it
is then allowed to express in a host cell. Thereafter, the secreted
supernatant is recovered and is then purified, so as to obtain a
human antibody. Alternatively, such a human antibody can also be
obtained by allowing VH and VL to express in a single vector
(tandem type). These methods are well known, and can be carried out
with reference to WO92/01047, WO92/20791, WO93/06213, WO93/11236,
WO93/19172, WO95/01438, WO95/15388, WO97/10354, etc.
[0067] Specifically, DNA encoding VH is ligated to another DNA
molecule encoding a heavy chain constant region (CH1, CH2 and CH3),
so as to obtain a full-length heavy chain gene. The sequence of a
human heavy chain constant region gene is known in the present
technical field (for example, Kabat, E. A. et al., (1991) Sequences
of Proteins of Immunological Interest, 5.sup.th edition, U. S.
Department of Health and Human Services, NIH Publication No.
91-3242), and a DNA fragment including such a region can be
obtained by standard PCR amplification. The heavy chain constant
region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA,
IgE, IgM or IgD. The most preferred constant region is that of IgG1
or IgG2. The constant region sequence of IgG1 may include any given
various alleles or allotypes known to be generated among different
individuals, such as Gm (1), Gm (2), Gm (3) or Gm (17). These
allotypes correspond to a substitution of amino acids
naturally-occurring in the constant region of IgG1.
[0068] DNA encoding VL is ligated to another DNA molecule encoding
the light chain constant region CL, so as to obtain a full-length L
chain gene (and a Fab light chain gene). The sequence of a human
light chain constant region gene is known in the present technical
field (for example, Kabat, E. A. et al., (1991) Sequences of
Proteins of Immunological Interest, 5.sup.th edition, U. S.
Department of Health and Human Services, NIH Publication No.
91-3242), and a DNA fragment including such a region can be
obtained by standard PCR amplification. The light chain constant
region may be the constant region of .kappa. or .lamda.. The
.kappa. constant region may include any given various alleles known
to be generated among different individuals, such as Inv (1), Inv
(2) or Inv (3). The .lamda. constant region may be derived from any
one of the three .lamda. genes.
[0069] The thus obtained DNA encoding an H chain or L chain is
inserted into a vector to produce an expression vector, and the
produced expression vector is then allowed to express in a host
cell. Thereafter, the secreted supernatant is recovered and
purified to obtain a human antibody. Examples of the expression
vector include a plasmid, retrovirus, adenovirus, adeno-associated
virus (AAV), plant viruses such as cauliflower mosaic virus or
tobacco mosaic virus, a cosmid, YAC, and EBV-derived episome. An
expression vector and an expression regulatory sequence are
selected, so that they are suitable for a host cell used for
expression. An antibody light chain gene and an antibody heavy
chain gene can be inserted into different vectors, or the two genes
can also be inserted into a single expression vector. An antibody
gene is inserted into an expression vector by a standard method
(for example, ligation of a complementary restriction site on an
antibody gene fragment to a vector, or blunt-ended ligation applied
when no restriction sites are present).
[0070] A preferred vector encodes a functionally completed human CH
or CL immunoglobulin sequence having a suitable restriction site,
which has been produced by an engineering approach such that any
given VH or VL sequence can be easily inserted and then expressed
therein, as described above. In such a vector, splicing generally
takes place between a splice donor site in the inserted J region
and a splice acceptor site preceding a human C domain, or such
splicing also takes place in a splice region existing in a human CH
exon. Polyadenylation and transcription termination take place in a
natural chromosomal site downstream of a coding region. A
recombinant expression vector can also encode a signal peptide that
promotes the secretion of an antibody chain derived from a host
cell. An antibody chain gene can be cloned into a vector, such that
a signal peptide can be ligated in-frame to the amino terminus of
an immunoglobulin chain. The signal peptide may be either an
immunoglobulin signal peptide or a heterogeneous signal peptide
(namely, it may be a non-immunoglobulin protein-derived signal
peptide).
[0071] An expression vector used for the antibody of the present
invention may also have sequences such as a sequence for regulating
replication of the vector in a host cell (e.g. a replication
origin) or a selective marker gene sequence, in addition to an
antibody gene and a regulatory sequence. The selective marker gene
promotes selection of a host cell into which a vector has been
introduced. For instance, the selective marker generally imparts
resistance to drugs such as G418, hygromycin or methotrexate to a
host cell into which the vector has been introduced. Preferred
selective marker genes include a dihydrofolate reductase (DHFR)
gene (used in methotrexate selection/amplification as a dhfr-host
cell), a neomycin phosphotransferase gene (used in G418 selection),
and a glutamate synthase gene.
[0072] A host cell is transformed with an antibody gene expression
vector produced by the above-described method. Any type of cell may
be used as a host cell, as long as it can produce the antibody of
the present invention. Examples of such a host cell include
bacteria, yeast, animal cells, insect cells, and plant cells. Among
these cells, animal cells are preferable. Examples of the animal
cells include Chinese hamster ovary cells CHO/dhfr(-) and CHO/DG44,
monkey-derived cells COS (A. Wright & S. L. Morrison, J.
Immunol. 160, 3393-3402 (1998)), and SP2/O cells (mouse myeloma)
(K. Motmans et al., Eur. J. Cancer Prev. 5, 512-5199 (1996), R. P.
Junghans et al., Cancer Res. 50, 1495-1502 (1990)). For
transformation, a lipofectin method (R. W. Malone et al., Proc.
NAML. Acad. Sci. USA 86, 6007 (1989), P. L. Feigner et al., Proc.
NAML. Acad. Sci. USA 84, 7413 (1987)), an electroporation method, a
calcium phosphate method (F. L. Graham & A. J. van der Eb,
Virology 52,456-467 (1973)), a DEAE-Dextran method, and the like
are preferably applied.
[0073] A transformant is cultured, and a human antibody is then
separated from the cells of the transformant or a culture medium
thereof. For separation/purification of the antibody, methods such
as centrifugation, ammonium sulfate fractionation, salting-out,
ultrafiltration, affinity chromatography, ion exchange
chromatography and gel filtration chromatography can be used by
appropriately combining them.
Antigen-Binding Fragments
[0074] An antigen-binding fragment can be produced based on the
antibody of the present invention, or based on the sequence
information of a gene encoding the antibody of the present
invention. Examples of the antigen-binding fragment include Fab,
Fab', F(ab').sub.2, a single-chain antibody (scFv), a dimerized V
region (Diabody), a disulfide stabilized V region (dsFv), and a
peptide comprising a complementarity-determining region (CDR)
derived from an antibody.
[0075] Fab is obtained by digesting IgG by papain in the presence
of cysteine. It is an antibody fragment with a molecular weight of
approximately 50,000, which is constituted with L chain and H chain
variable regions, and an H chain fragment consisting of a CH1
domain and a portion of a hinge region. In the present invention,
the above-described antibody can be obtained by papain digestion.
In addition, Fab can also be prepared by incorporating DNA encoding
a portion of the H chain and the L chain of the above-described
antibody into a suitable vector, then performing transformation
with the resulting vector, and then obtaining it from the
transformant.
[0076] Fab' is an antibody fragment with a molecular weight of
approximately 50,000, which is obtained by cleaving a disulfide
bond between the H chains of the below-mentioned F(ab').sub.2. In
the present invention, Fab' can be obtained by digesting the
above-described antibody by pepsin, and then cleaving a disulfide
bond with a reducing agent. In addition, as with Fab, Fab' can also
be prepared by genetic engineering using DNA encoding the Fab'.
[0077] F(ab').sub.2 is obtained by digesting IgG by pepsin, and is
an antibody fragment with a molecular weight of approximately
100,000, which is obtained by binding, via a disulfide bond, one
fragment (Fab') constituted with L chain and H chain variable
regions and an H chain fragment consisting of a CH1 domain and a
portion of a hinge region, to the other fragment (Fab'). In the
present invention, F(ab').sub.2 can be obtained by digesting the
above-described antibody by pepsin. In addition, as with Fab,
F(ab').sub.2 can also be prepared by genetic engineering using DNA
encoding the F(ab').sub.2.
[0078] scFv is an antibody fragment obtained by ligating the
C-terminus of one chain of Fv consisting of an H chain variable
region and an L chain variable region to the N-terminus of the
other chain thereof, using a suitable peptide linker, so as to form
a single chain. (GGGGS).sub.3 having high flexibility can be used,
for example, as such a peptide linker. For instance, DNA encoding
the H chain variable region and L chain variable region of the
above-described antibody and DNA encoding a peptide linker are used
to construct DNA encoding a scFv antibody, and the thus constructed
DNA is then incorporated into a suitable vector. Thereafter, scFv
can be prepared from a transformant obtained by transformation with
the aforementioned vector.
[0079] The dimerized V region (Diabody) is a dimer obtained by
binding two fragments (e.g., scFv, etc.) to each other, each of
which has been formed by binding a variable region to another
variable region via a linker or the like. In general, Diabody
comprises two VL regions and two VH regions. The bond between the
fragments constituting the Diabody may be either a non-covalent
bond or a covalent bond. It is preferably a non-covalent bond.
[0080] dsFv is a Fv fragment obtained by introducing a Cys residue
into a suitable site in each of an H chain variable region and an L
chain variable region, and then stabilizing the H chain variable
region and the L chain variable region by a disulfide bond. The
site in each chain, into which the Cys residue is to be introduced,
can be determined based on a conformation predicted by molecular
modeling. In the present invention, for example, a conformation is
predicted from the amino acid sequences of the H chain variable
region and L chain variable region of the above-described antibody,
and DNA encoding each of the H chain variable region and the L
chain variable region, into which a mutation has been introduced
based on such prediction, is then constructed. The thus constructed
DNA is incorporated into a suitable vector. Thereafter, dsFv can be
then prepared from a transformant obtained by transformation with
the aforementioned vector.
[0081] Further, it is also possible to ligate scFv antibody, dcFv
antibody or the like using a suitable linker, or to fuse such an
antibody fragment with streptavidin, so as to multimerize the
antibody fragment.
Pharmaceutical Agent
[0082] According to the present invention, an inhibitor of cancer
cell adhesion, which comprises the antibody of the present
invention, and a pharmaceutical composition, which comprises the
antibody of the present invention, are provided. In one embodiment,
the present invention relates to the treatment of refractory
leukemia, but is not limited thereto. Diseases caused by
overexpression of an ITGA6B4 complex, other than refractory
leukemia, can also be treated using the antibody of the present
invention. Preferably, the therapeutic target is refractory
leukemia. However, solid cancer (e.g. lung cancer, colon cancer,
stomach cancer, bladder cancer, pancreatic cancer, prostate cancer,
hepatic cancer, cervical cancer, uterine cancer, ovarian cancer,
breast cancer, head and neck cancer, skin cancer, etc.), blood
cancer (e.g. leukemia, lymphoma, myeloma, etc.), and the like are
also included in the therapeutic target.
[0083] The inhibitor of cancer cell adhesion and the pharmaceutical
composition, each of which comprises the antibody of the present
invention, may comprise a pharmaceutically acceptable carrier, an
excipient, a diluent, and the like, as appropriate. The inhibitor
of cancer cell adhesion and the pharmaceutical composition of the
present invention can be each formulated, for example, in the form
of an injection. The applied doses of the inhibitor of cancer cell
adhesion and the pharmaceutical composition of the present
invention depend on the symptom degree, age and body weight of a
patient, an administration method, and the like, and the weight of
an antibody as an active ingredient is generally in the range of
approximately 10 ng to approximately 100 mg/kg of body weight per
day.
Detection of Cells
[0084] The antibody of the present invention, which has bound to
cells, can be detected by flow cytometry, ELISA, or a combination
thereof. As a device for detecting or quantifying cells, to which
the antibody of the present invention has bound, a flow cytometer
(FACS: Fluorescence Activated Cell Sorter) is preferable. Methods
of using other measurement devices capable of measuring circulating
tumor cells (CTC) may also be applied. Most preferably, cells, to
which the antibody of the present invention has bound, can be
measured using a flow cytometer. In this case, the antibody of the
present invention has been preferably labeled with fluorescent dye.
Blood cells obtained from a blood specimen are allowed to come into
contact with the antibody of the present invention, which has been
fluorescently labeled, and thereafter, the cells can be analyzed
using a flow cytometer.
[0085] The present invention will be described more in detail in
the following examples. However, these examples are not intended to
limit the scope of the present invention.
EXAMPLES
Example 1
Screening for Phage Antibody Using Cancer Cell Line
(1) Screening for Phage Antibody Bound to Cancer Cells (AML Line
USCD-AML1)
[0086] USCD-AML1 cells (Oval et al., Blood 76: 1369-1374 (1990);
Taetle et al., Cancer Res. 53: 3386-3393 (1993); Hu et al.,
Leukemia 10: 1025-1040 (1996)) were cultured by any given method.
The cells were recovered and were then washed with cooled PBS.
Thereafter, a human antibody phage library of 1.times.10.sup.13 cfu
(see JP Patent Publication (Kokai) No. 2005-185281 A, WO
2008/007648, and WO 2006/090750) was mixed with the resulting
cells, and a reaction solution (1% BSA, 0.1% NaN3, and MEM) was
then added thereto to a final volume of 1.6 mL. The obtained
mixture was slowly rotated at 4.degree. C. for 4 hours for
performing a reaction. After completion of the reaction, the
reaction solution was divided into two aliquots, and each aliquot
was then layered on 0.6 mL of an organic solution (dibutyl
phthalate and cycloheximide (9:1)) that had previously been
prepared, and the thus obtained mixture was then centrifuged (3000
rpm) for 2 minutes using a microcentrifuge. Thereafter, the
supernatant was discarded, and cells precipitated at the bottom of
the tube were suspended in 0.7 mL of 1% BSA/MEM. Then, the obtained
suspension was further layered on 0.7 mL of an organic solvent.
Centrifugation was carried out in the same manner as described
above, the supernatant was then discarded, and the cells were then
suspended in 0.3 mL of PBS, followed by freezing with liquid
nitrogen.
[0087] The frozen cells were thawed at 37.degree. C., and were then
infected with 20 mL of Escherichia coli DH12S (OD0.5) for 1 hour.
The phage-infected Escherichia coli was placed in 600 mL of a
2.times.YTGA medium (2.times.YT, 200 .mu.g/mL ampicisulfate, and 1%
glucose), and it was then cultured at 30.degree. C. overnight.
Thereafter, 10 mL of the culture was placed in 200 mL of a
2.times.YTA medium (2.times.YT and 200 .mu.g/mL ampicisulfate), and
it was then cultured at 37.degree. C. for 1.5 hours. Then,
1.times.10.sup.11 helper phage KO7 was added to the culture, and
the obtained mixture was further cultured at 37.degree. C. for 1
hour. Subsequently, 800 mL of a 2.times.YTGAK medium (2.times.YT,
200 .mu.g/mL ampicisulfate, 0.05% glucose, and 50 .mu.g/mL
kanamycin) was added to the culture, and the obtained mixture was
then cultured at 30.degree. C. overnight. Thereafter, the
supernatant was recovered by centrifugation (8000 rpm) for 10
minutes. To the recovered supernatant, 200 mL of a PEG solution
(20% polyethylene glycol 6000 and 2.5M NaCl) was added, and the
obtained mixture was fully stirred. Thereafter, the reaction
mixture was subjected to centrifugation (8000 rpm) for 10 minutes
to precipitate phages. The phages were suspended in 10 mL of PBS.
The obtained solution was defined as phages obtained from the
1.sup.st screening.
[0088] Subsequently, the 2.sup.nd screening was carried out. The
cultured cells (2.times.10.sup.7) were mixed with the phages from
the 1.sup.st screening (1.times.10.sup.10), and a reaction solution
(1% BSA, 0.1% NaN3, and MEM) was added to the mixture to a final
volume of 0.8 mL. Thereafter, the same operations as those in the
aforementioned 1.sup.st screening were carried out, so as to obtain
phages from the 2.sup.nd screening.
[0089] The 3.sup.rd screening was carried out using the phages
(1.times.10.sup.9) obtained from the 2.sup.nd screening in the same
manner as described above.
(2) Analysis of Phage Antibodies
[0090] The phages obtained from the 3.sup.rd screening were
recovered, and the DNA sequences thereof were then analyzed by the
existing method. Incomplete antibodies comprising deletions in the
regions or antibodies having overlapping sequences were removed, so
that phage antibodies each having an independent antibody sequence
could be obtained (see Japanese Patent No. 4870348).
Example 2
Screening for Positive Phages by ELISA
[0091] Using a purified soluble human ITGA6B4 antigen (Recombinant
Human Integrin alpha 6 (.times.1) beta 4, R & D), the
reactivity of antigen-antibody was examined by ELISA. Specifically,
the concentration of the soluble ITGA6B4 antigen was adjusted to be
10 .mu.g/mL with PBS, and it was then added to Immuno Module/Strip
Plates (NUNC) in an amount of 50 .mu.L/well. It was left at rest at
37.degree. C. for 2 hours. Thereafter, the soluble ITGA6B4 antigen
was discarded, and a blocking solution (5% skimmed milk/0.05%
NaN3/PBS) was added thereto in an amount of 200 .mu.L/well,
followed by performing blocking at 37.degree. C. for 2 hours.
Thereafter, the blocking solution was removed, and the residue was
then washed with PBS. The culture supernatant of the
above-mentioned phage from the second screening was added to each
well in an amount of 100 .mu.L/well, and it was then reacted at
37.degree. C. for 1 hour. The resultant was washed with PBS five
times, and 1 .mu.g/mL Rabbit anti-cp3 that had been diluted with
PBS/0.05% Tween 20 was then added to the resultant in an amount of
100 .mu.L/well. The thus obtained mixture was reacted at 37.degree.
C. for 1 hour. The resultant was washed with PBS five times, and
anti-Rabbit IgG (H+L)-HRP that had been 2000 times diluted with
PBS/0.05% Tween 20 was further added to the resultant in an amount
of 100 .mu.L/well. The thus obtained mixture was reacted at
37.degree. C. for 1 hour. The resultant was washed with PBS five
times, and OPD in a 0.1 M citrate phosphate buffer (pH 5.1)+0.01%
H.sub.2O.sub.2 was then added thereto in an amount of 100
.mu.L/well. The obtained mixture was reacted at room temperature
for 5 minutes. Thereafter, 2NH.sub.2SO.sub.2 was added to the
reaction solution in an amount of 100 .mu.L/well, so as to
terminate the coloring reaction. Subsequently, the absorbance at
492 nm was measured using SPECTRA max340PC (Molecular Devices). As
a result, the DNA sequences of phage clones exhibiting a
significant positive reaction with the soluble ITGA6B4 antigen were
analyzed, and their CDR sequences were each confirmed, and those
having an independent sequence were then classified. As a result,
five antibodies having a particularly strong reactivity were
selected, and they were referred to as PPMXITG-001 to -005.
[0092] With regard to PPMMTG-001 to -005, their heavy chain first
complementarity-determining region (VH CDR1), heavy chain second
complementarity-determining region (VH CDR2), heavy chain third
complementarity-determining region (VH CDR3), light chain first
complementarity-determining region (VL CDR1), light chain second
complementarity-determining region (VL CDR2), and light chain third
complementarity-determining region (VL CDR3) are shown below.
TABLE-US-00001 TABLE 1 VH CDR1 VH CDR2 VH CDR3 VL CDR1 VL CDR2 VL
CDR3 PPMXITG-001 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO:
4 SEQ ID NO: 5 SEQ ID NO: 6 PPMXITG-002 SEQ ID NO: 7 SEQ ID NO: 8
SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 PPMXITG-003
SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO:
17 SEQ ID NO: 18 PPMXITG-004 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO:
21 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 PPMXITG-005 SEQ ID NO:
25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID
NO: 30
Example 3
Production of IgG Antibody from Phage Antibody (scFv)
(1) Production of Plasmid Expressing ITGA6B4 IgG Antibody
[0093] Formation of an IgG antibody from a phage antibody will be
explained below, giving formation of an IgG antibody from ITGA6B4
as an example.
[0094] The VII and VL genes of the phage antibody (scFv) of ITGA6B4
are aligned in the order of VH-VL, and the VH and the VL are
connected with each other via a linker having the following
sequence, so as to have the structure of scFv.
<Linker Sequence>
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID
NO: 31)
[0095] A human germline gene assumed to be used in the VII and VL
of ITGA6B4 was searched on IMGT (*) (* IMGT: http://www.imgt.org),
and a phage antibody was processed into an IgG antibody thereof;
with reference to the search results of IMGT. The VH of ITGA6B4 was
connected with the constant region of human IgG 1. With regard to
the VL of ITGA6B4, the H chain and L chain genes, to which NheI was
added to the 5' side thereof and EcoRI was added to the 3' side
thereof, were totally synthesized by GenScript. The thus
synthesized heavy chain and light chain genes were each
incorporated into different expression vectors. That is to say,
artificially synthesized genes of the H chain and the L chain were
each cleaved with NheI and EcoRI, and the cleavages were then
incorporated into the NheI and EcoRI sites of the expression vector
pCAGGS, so as to obtain an anti-ITGA6B4 antibody H chain expression
vector and an anti-ITGA6B4 L chain expression vector.
[0096] Expression vectors for other antibodies were produced by the
same method as described above.
(2) Transient Expression of IgG Antibody
[0097] FreeStyle (Life Technologies) was used for transient
expression of an IgG antibody. 293-F (Life Technologies) used as
floating cells for gene transfection was subcultured the day before
transfection. On the day of transfection, 400 mL of a cell
suspension whose cell density had been adjusted to be
1.times.10.sup.6 cells/mL was prepared for the expression of one
type of antibody. Solution I was prepared by suspending a total of
200 .mu.g of plasmid consisting of 100 .mu.g of an antibody heavy
chain expression vector and 100 .mu.g of an antibody light chain
expression vector in OptiPro SFM. Subsequently, 200 .mu.L of MAX
reagent was added to 8 mL of OptiPRO (Solution II). Solution (I)
was mixed with Solution (II), and the thus mixed solution was then
left at rest at room temperature for 10 to 20 minutes. A total of
16 mL of the reaction solution was added to 400 mL of a 293
expression medium, in which the 293-F cells had been suspended, and
the obtained mixture was then cultured at 37.degree. C. in 8%
CO.sub.2 for 6 to 7 days, using a cell culture shaker TAITEC
BioShaker BR-43FL. After 6 to 7 days of the culture, a culture
supernatant containing each recombinant antibody was recovered, and
this antibody was used as a material for purification.
(3) Purification of IgG Antibody
[0098] The IgG antibody protein contained in the culture
supernatant, which had been expressed above, was purified employing
an Ab-Capcher ExTra (ProteNova) affinity column using AKTAprime.
The obtained peak fraction was subjected to gel filtration using a
Sephacryl S-300 column that had been equilibrated with Dulbecco's
PBS as a solvent, so as to further purify it. The purified IgG
antibody protein was quantified using an absorption coefficient.
The absorption coefficient of the IgG antibody was calculated
employing EXPASY ProtParam (http://web.expasy.org/protparam/),
using total amino acid sequences of individual antibodies.
(4) Quantification of Anti-ITGA6B4 IgG Antibody by Enzyme-Linked
Immunosorbent Assay (ELISA)
[0099] The concentration of an antibody contained in the culture
supernatant of anti-ITGA6B4 IgG antibody-producing cells, which had
been expressed and purified as described above, or the
concentration of a purified antibody was quantified based on
absorbance, and was also quantified by enzyme-linked immunosorbent
assay (ELISA). As a solid-phase antibody, goat anti-human IgG (H+L)
(which had previously been absorbed against mouse, rabbit, bovine,
and mouse IgG) (COMSO BIO: American Qualex International, Inc.;
AQI, Cat. No. A-110UD) was added in an amount of 100 .mu.l/well
(concentration: 5 .mu.g/mL) to a plate, and it was then left at
rest at 4.degree. C. over a day and a night. Subsequently, Block
Ace was added in an amount of 200 .mu.L/well to the plate to block
the antibody at room temperature for 1 hour. Thereafter, the
antibody as a sample was subjected to serial dilution, and it was
then added to each well, followed by incubation for 1 hour for
performing a reaction. The reaction product was washed with PBST
(0.05% Tween 20 and PBS) five times, and then, a detection antibody
solution prepared by 10,000 times diluting goat anti-human IgG
(H+L) (absorbed against mouse, rabbit, bovine, and mouse IgG)--HRP
(COSMO BIO: AQI, Cat. A-110PD) with PBST was added in an amount of
100 .mu.L/well to the resultant. The obtained mixture was incubated
for 1 hour, and was then washed with PBST five times. Thereafter, a
substrate buffer TMB was added in an amount of 100 .mu.L/well to
the resultant. The obtained mixture was incubated at room
temperature in a dark place for 15 minutes, and a reaction
termination solution was then added thereto in an amount of 100
.mu.L/well so as to terminate the reaction. Thereafter, the
absorbance at 450 nm was measured. Using purified human IgG as a
standard product, a calibration curve was produced, and the
concentration of a human antibody was calculated using this
calibration curve.
Example 4
Reactivity of Anti-ITGA6B4 IgG Antibody with Cell Lines
[0100] The reactivity of the anti-ITGA6BA IgG antibody was examined
using an ITGA6B4-expressing cell line, AML, cell line USCD-AML1.
USCD-AML1 cells were recovered by centrifugation. The recovered
cells were washed with PBS once, and the resulting cells were
suspended in FACS Buffer (PBS containing 1% BSA, 2 mM EDTA, and
0.1% NaN3), resulting in a cell density of 1.times.10.sup.6
cells/mL. 100 .mu.L of this cell suspension was dispensed into a
96-well V bottom plate (Costar 3897). Thereafter, each ITGA6B4 IgG
antibody was adjusted to be 0.02 to 2 .mu.g/mL with FACS Buffer,
and 100 .mu.L of the prepared antibody solution was then added to
the cells. The obtained mixture was incubated at 4.degree. C. for 1
hour. Thereafter, the resulting cells were washed with FACS Buffer,
and 100 .mu.L of Alexa488-anti-human IgG (Invitrogen) solution that
had been 750 times diluted with FACS Buffer was then added to the
cells. The thus obtained mixture was further incubated at 4.degree.
C. for 1 hour. The resultant was washed by centrifugation with FACS
Buffer twice, and was then equipped into HTS of FACS Calibur (BD),
and the fluorescence intensity of FL1 in each well was measured. As
a result, all of the antibodies (a: 10 ng/mL; b: 100 ng/mL; c: 1
.mu.g/mL) exhibited strong reactivity with USCD-AML1. In addition,
in order to examine whether the reactivity with a purified ITGA6B4
antigen did not disappear, ELISA was carried out by the same
procedures as those of Example 2, and the reactivity was confirmed.
An example thereof is shown in FIG. 4. As shown in FIG. 4, a clear
positive reaction was obtained. As negative control antibodies, an
anti-habu venom human IgG antibody, which had been produced by the
same procedures as those for the concerned antibody, and a
detection antibody only (described as "PBS), were used. A
representative example of the antibody described as "anti-ITGA6BA
antibody" is PPMXITG-001.
Example 5
Immunoprecipitation Using Anti-ITGA6BA Antibody and High Expression
Cell Line
(1) Preparation of Solid-Phased Antibody Used for
Immunoprecipitation
[0101] First, the above-produced anti-ITGA6B4 antibody solution was
dialyzed against a coupling buffer solution (0.1M NaHCO3-NaOH,
pH9). Specifically, the antibody solution was enclosed within a
dialysis membrane (Snake Skin Pleated Dialysis Tubing 10,000 MWCO),
and this membrane was then immersed in 1.5 L of a coupling buffer
solution (0.1M NaHCO3-NaOH, pH9). The solution was stirred at
4.degree. C. for 2 to 3 hours using a stirrer, and the buffer
solution was then exchanged with a fresh one, followed by
performing a further dialysis for 2 to 3 hours. Thereafter, the
buffer solution was exchanged with a fresh one again, and the
dialysis was then carried out over a day and a night.
[0102] Subsequently, activated CNBr-activated Sepharose 4B to be
used for solidification was prepared. Specifically, CNBr-activated
Sepharose 4B manufactured by Amersham Biosciences was swollen with
1 mM HCl, and was then aspirated using an aspirator. To the
resultant, 50 ml of a coupling buffer solution was added, and
thereafter, the mixture was stirred and was then aspirated using an
aspirator. While the mixture was aspirated, a coupling buffer
solution was further added thereto.
[0103] Solidification of the antibody was carried out as follows.
Specifically, 1 ml of activator gel was added to 5 mg of the
antibody in 10 ml of the solution, and the obtained mixture was
then reacted at room temperature for 2 hours. After completion of
the reaction, the gel was transferred into a column, and was then
washed with 1 ml of a coupling buffer solution ten times.
Thereafter, the presence or absence of an unreacted antibody was
confirmed by O.D. measurement. The solid-phased gel was replaced
with 5 ml of a 0.2 M Glycine-NaOH solution (pH 8) twice, and 5 ml
of the same solution as described above was then added thereto. The
mixed solution was left at rest at room temperature for 2 hours,
and the solution was then subjected to natural dripping. After
that, 5 ml of 0.2 M Glycine-HCl (pH 3) was added thereto for
replacement, and 5 ml of the same solution as described above was
further added thereto. The obtained solution was left at rest for 5
minutes, followed by natural dripping Finally, the column was
replaced with 20 ml of PBS, followed by natural dripping, and then,
1% NP40, a protease inhibitor, and 0.05% NaN.sub.3/PBS were added
to the resulting solution, so as to recover gel.
(2) Labeling of Protein on Cell Membrane with Biotin and Production
of Cell Lysate
[0104] A cultured hepatic cancer cell line was labeled with biotin
as follows. Specifically, the cultured cells PC 14, which had been
cultured to a cell count of approximately 10.sup.7 to 10.sup.8,
were washed with PBS twice, and Collagenase I (manufactured by
GIBCO), which had been adjusted to a concentration of 5 mg/ml with
a cell dissociation buffer (manufactured by GIBCO), was then added
to the resulting cells. The obtained mixture was reacted at
37.degree. C. in a CO.sub.2 incubator, so that the cells were
released. Thereafter, the cells were recovered with a medium and
were then washed with PBS(-) twice, and the number of cells was
calculated using a hemocytometer. The cells were suspended in
PBS(-), resulting in a concentration of approximately
5.times.10.sup.7/ml. To this suspension, EZ-Link
Sulfo-NHS-LC-Biotinylation Kit (PIERCE), which had been adjusted to
1 mg/ml with PBS, was added in an equal amount. The obtained
mixture was left at rest at room temperature for 30 minutes, and
was then washed with PBS twice.
[0105] A cell lysate was prepared from the biotin-labeled cells as
follows. Specifically, 4 ml of a lysis buffer (a 1% NP40/detergent
base solution, wherein the detergent base solution comprised 20 mM
HEPES (pH 8.0), 140 mM NaCl, and a protease inhibitor) was added to
the above-described biotin-labeled cells, so that the cells were
suspended therein. The obtained suspension was placed in a Dounce
homogenizer that had been cooled, and it was then homogenized.
Thereafter, to the resulting solution, a detergent mix solution (1%
NP40, tritonX-100,b-D-Maltoside, n-Octyl b-D-Glucoside, n-Octyl
b-D-Maltoside, n-Decyl b-D-Maltoside, and deoxycholic acid, in each
amount of 0.5%/detergent base solution) was added in a half amount
(2 ml), and the obtained mixture was then subjected to rotation
mixing at 4.degree. C. for 4 hours. Thereafter, the mixed solution
was centrifuged at 100,000 rpm for 30 minutes, and was then
filtrated through a 0.22-.mu.m filter MILLEX-GP.
(3) Immunoprecipitation Reaction
[0106] First, a solid-phased antibody (hereinafter referred to as
"antibody beads") corresponding to approximately 60 .mu.l (which
was approximately 150 .mu.l of solution) was placed in a 2-ml tube,
and 4 mM biotin was then added in an amount of 1/10 volume
(approximately 15 .mu.l) to the tube. To this mixture, a lysate
(600 .mu.l) corresponding to a half culture dish that had been
mixed with 60 .mu.l of a biotin solution was added, and the thus
obtained mixture was then reacted at 4.degree. C. for several
hours, while stirring. Thereafter, the tube was centrifuged (5500
g, 1 minute, 4.degree. C.), and a supernatant was then removed. To
the residue, 800 .mu.l of a biotin/lysis-T buffer for washing (0.5
mM biotin, 0.1% Tween 20/PBS) was added, and the obtained mixture
was then subjected to inversion mixing two or three times. The tube
was centrifuged (5500 g, 1 minute, 4.degree. C.), and a supernatant
was then removed. This washing operation was carried out again, and
30 .mu.l of a citric acid solution for elution (50 mM citric acid
(pH 2.5)) was then added to the antibody beads, followed by
stirring. Thereafter, the tube was centrifuged (5500 g, 1 minute,
4.degree. C.), and a supernatant was then recovered. To the
remaining antibody beads, 30 .mu.l of a citric acid solution for
elution was added again, followed by stirring, the obtained mixture
was then centrifuged (5500 g, 1 minute, 4.degree. C.), and a
supernatant was recovered. This elution operation was further
carried out repeatedly three times, and a sample solution was then
recovered. To the recovered sample solution, 3 M Tris was added for
neutralization. The obtained sample was electrophoresed by
SDS-PAGE, and bands were then confirmed by silver staining. This
sample was also subjected to Western-blot using streptavidin-HRP
(Anti-Streptavidin, IgG Fraction, Conjugated to Peroxidase CORTEX
biochem), so that the biotinylated membrane protein bands were
detected. As a result, as shown in FIG. 1, two bands were detected.
The anti-ITGA6BA antibodies 1, 2 and 3 shown in FIG. 1 correspond
to PPMXITG-001, 004 and 005, respectively.
Example 6
Mass Spectrometry Performed on Immunoprecipitated Bands
(1) Trypsin Digestion in Gel
[0107] A portion corresponding to the membrane protein detected in
the above-described Example 5 was digested with trypsin in gel, so
that a peptide was recovered. An SDS polyacrylamide gel
electrophoresis was carried out according to an ordinary method,
and a band obtained by staining with Coomassie brilliant blue was
cut out. This band was immersed in a 200 mM ammonium
bicarbonate-50% acetonitrile solution, and it was then shaken at
37.degree. C. for 45 minutes. Thereafter, the solution was
discarded, and the same operation as described above was repeated
twice, so as to remove Coomassie brilliant blue. The resulting gel
was dried under reduced pressure, and trypsin (20 .mu.g/ml)
dissolved in 40 mM ammonium bicarbonate (pH 8.1)-10% acetonitrile
was then added to the gel in an amount of 4 .mu.l per unit area
(mm2) of a gel slice. The gel was left at room temperature for 1
hour, so that it was fully swollen. To the gel, a trypsin solution
was added in an amount of 2.5 times the previously added trypsin
solution, and the obtained mixture was then left at rest at
37.degree. C. for 18 hours. Thereafter, the resultant was filtrated
with a tube equipped with a filter having a pore size of 0.22
.mu.m, so as to recover a peptide generated as a result of the
cleavage of the antigen with trypsin.
(2) Identification of Antigen by Mass Spectrometry
[0108] The sample obtained by trypsin digestion in gel was
subjected to HPLC, which was connected with an electrospray
ionization ion trap quadrupole mass spectrometer. Individual
peptides, which were successively eluted from a reverse phase
chromatography column of the HPLC based on a difference in
hydrophobicity as a result of changes in a linear concentration
gradient of 0% to 80% acetonitrile comprising 0.1% TFA, were
ionized by an electrospray method. Thereafter, the mass of each
peptide was analyzed.
[0109] At the same time, the mass of the limited decomposition
product of each peptide generated as a result of collision of the
peptide with a helium atom placed on the midcourse of the flight
path of ions was analyzed. When one amino acid is dissociated by
limited decomposition, a smaller ion by the mass of the dissociated
amino acid is observed. Thus, the type of the dissociated amino
acid can be identified based on the mass difference. When another
amino acid is dissociated, a smaller ion by the mass of the
dissociated amino acid is observed. Thus, the type of the
dissociated amino acid can be identified based on the mass
difference. The internal amino acid sequence can be determined by
conducting the same experimental data analysis as described above.
A set of the obtained internal amino acid sequences was searched
against the published amino acid sequence database, so as to
identify the antigens. Consequently, it was demonstrated that the
two bands obtained in Example 5 were ITGA6 (FIG. 2) and ITGB4 (FIG.
3).
Example 7
Analysis of Adhesion-Inhibiting Ability Using Anti-ITGA6/B4
Antibody
[0110] The ability of the anti-ITGA6/B4 antibody of the present
invention to inhibit the adhesiveness of refractory leukemia cell
lines was analyzed. As cell lines used as analysis targets, three
cell lines, which highly express EVI1 as a marker molecule for
refractory leukemia, were selected (UCSD/AML1, Kasumi-3, MOLM1, and
HNT34).
[0111] A plate was coated with Matrigel (BD Bioscience) as a
basement membrane matrix, so that an easy adhesive environment that
was similar to a bone marrow niche was produced. A comparison was
made among the cell lines, in terms of the adhesiveness of the
cells treated with antibodies under the aforementioned conditions.
As a result, it has become clear that the adhesiveness of the cells
is decreased by addition of any of anti-ITGA6/B4 antibodies
(PPMXITG-001, 004, and 005) (FIG. 6).
Example 8
Experiment for Examining Effects Obtained by Combined Use of the
Antibody of the Present Invention with Anticancer Agent
[0112] It has been anticipated that the anti-ITGA6/B4 antibody of
the present invention enhances the effects of an anticancer agent.
Hence, using the refractory leukemia cell lines UCSD/AML1, Kasumi3,
MOLM1 and HNT34, the influence obtained by the combined use of the
present antibody with anticancer agents commonly used for the
treatment of leukemia, namely, AraC (cytarabine), DXR (doxorubicin)
and VP16 (etoposide) was analyzed.
[0113] A comparison was made among the leukemia cell lines, in
terms of their survival rate under conditions consisting of a
single use of anticancer agent and the combined use of an
anticancer agent and an antibody. As a result, it was confirmed
that IC50 was clearly decreased by addition of the antibodies
(PPMXITG-001, 004, and 005) (FIGS. 8-1 and 8-2). It is to be noted
that FIG. 8-1 shows an example of the tests of examining combined
effects, which were performed using DXR (doxorubicin) and AML1
cells.
[0114] From the obtained results, it was considered that the
anti-ITGA6/B4 antibody has an action to enhance the effects of
anticancer agents, and it was suggested that, by using the
anti-ITGA6/B4 antibody, high effects can be obtained with the use
of a smaller amount of anticancer agent. Moreover, it could be
anticipated that side effects would be reduced by decreasing the
effective dose of the anticancer agent.
REFERENCE
[0115] (1) Alkylating agents: ifosfamide, nimustine hydrochloride,
procarbazine hydrochloride, cyclophosphamide, dacarbazine,
thiotepa, etc. (2) Metabolic antagonists: cytarabine, enocitabine,
gemcitabine hydrochloride, carmofur, tegafur, fluorouracil, etc.
(3) Plant alkaloids: etoposide, irinotecan, docetaxel, paclitaxel,
vincristine, vindesine, vinblastine, etc. (4) Anticancer
antibiotics: actinomycin D, idarubicin, doxorubicin, hydrarubicin,
mitoxantrone, mitomycin C, etc. (5) Platinum-based formulations:
carboplatin, cisplatin, nedaplatin, oxaliplatin, etc. Product names
used in the description and abbreviation corresponding thereto:
[0116] AraC (cytarabine): This anticancer agent is mainly used for
blood cancer. This is a first-line agent for acute myeloid
leukemia.
[0117] DXR (doxorubicin): This anticancer agent is mainly used for
lymphoma.
[0118] VP16 (etoposide): This anticancer agent is used for
malignant lymphoma and acute leukemia.
UCSD/AML1: A cell line with a karyotype of t(3;3), which has been
established in Taetel UCSD. It is derived from Caucasian. Kasumi3:
A cell line with a karyotype of t(3;7), which has been established
at Hiroshima University. It is derived from Japanese. MOLM1: A cell
line with karyotypes of t(9;22) and inv(3), which has been
established at Hayashibara Biochemical Lab., Inc. It is derived
from Japanese. HNT34: A cell line with a karyotype of t(3;3), which
has been established at Tokyo Musashino Hospital. It is derived
from Japanese.
Example 9
Analysis of Ability of Anti-ITGA6BA Antibody to Inhibit Cell
Adhesion of AML1 Cell Lines
[0119] An experiment was carried out to examine inhibition of cell
adhesion of the four AML cell lines UCSD/AML1, Kasumi3, MOLM1 and
HNT34. A plate was coated with Matrigel (BD Bioscience) as a
basement membrane matrix, so that an easy adhesive environment that
was similar to a bone marrow niche was produced. A comparison was
made among the cell lines, in terms of the adhesiveness of the
cells treated with antibodies.
[0120] Specifically, the anti-ITGA6B4 IgG antibody, which had been
selected in Example 2, and had been then produced and evaluated in
Examples 3 and 4, was used to 10000 cells/100 .mu.L. The antibody
was added to the cells to a final concentration of 0 to 10
.mu.g/mL, and the obtained mixture was then incubated at 4.degree.
C. for 30 minutes. Thereafter, the reaction mixture was washed with
RPMI 1640/10% FCS. Subsequently, the resultant was suspended in 100
.mu.L of RPMI 1640/10% FCS, and the suspension was then added to
each well of a 96-well plate coated with Matrigel (registered
trademark). It was incubated at 37.degree. C. for 2 hours, and was
then washed with PBS three times. 90 .mu.L of RPMI 1640/10% FCS and
10 .mu.L of Cell counting kit 8 (manufactured by Dojindo
Laboratories) were added to each well, and the mixture was then
incubated at 37.degree. C. for 30 minutes. Thereafter, measurement
was carried out at a wavelength of 450 nm, using a
spectrophotometer. As a control, an untreated cell line was
used.
[0121] FIG. 7 is a graph showing the results of the aforementioned
cell adhesion assay. As shown in FIG. 7, the adhesion ability of
AML1 cells (the ratio of the number of adhering cells to the total
number of cells) was significantly reduced by the addition of
PPMXITG-001, PPMXITG-004, or PPMMTG-005.
Example 10
Search of the Expression Level of ITGA6B4 in Leukemic Stem
Cells
[0122] The expression pattern of the mRNA of ITGA6/B4 in healthy
human subjects and leukemia patients was analyzed in accordance
with the method described in Patent Literature 1. Using blood
provided by refractory leukemia patients and volunteers, the
expression of ITGA4/B1 (VLA4) and ITGA6/B4 in a hematopoietic stem
cell fraction and in a leukemic stem cell fraction was confirmed.
As a result, the data shown in FIG. 9 were obtained. The "HSC
derived from healthy subjects" indicates a CD34+ fraction from
normal samples (Hematopoietic Stem Cell: HSC), the "HSC derived
from leukemia patients" indicates a CD34+ fraction from leukemia
patient samples, and the "LSC derived from leukemia patients"
indicates a CD34+CD38- fraction (leukemic stem cell fraction: LSC).
By this search, it was confirmed again that the expression of ITGA6
and B4 is particularly high in the leukemic stem cells of leukemia
patients, and thus that ITGA6 and B4 are useful as therapeutic
targets.
Example 11
Evaluation of Reactivity of Antibody by Introduction of shRNA of
ITGA6 and ITGB4 Genes
[0123] In order to confirm that the antibody of the present
invention reacts with an intact ITGA6B4 complex which is expressed
on a cancer cell membrane, the reactivity of the antibody was
evaluated, using cells into which shRNA of ITGA6 and ITGB4 genes
had been introduced. Specifically, a UCSD/AML1 cell line was
transfected with each of shITGA6 and shITGB4, which are
small-hairpin RNAs (shRNAs) each having an activity of cleaving the
mRNAs of ITGA6 and ITGB4 genes, so as to obtain cell lines, in
which the expression of each gene was inhibited. Using the thus
obtained cell lines, a change in the reactivity of the antibody was
evaluated by FACS. Specific procedures for producing gene
expression-inhibited cell lines using shRNA, and the test of
examining the reactivity of the antibody by FACS were carried out
in accordance with the methods described in Patent Literature 1 and
Non Patent Literature 1.
[0124] Moreover, as commercially available ITGA6 and ITGB4 positive
control antibodies, an anti-human ITGA6 rat antibody (Santa Cruz)
and an anti-human ITGB4 mouse antibody (Millipore), which are
described in Non Patent Literature 1, were used.
[0125] As a result, the following data shown in FIG. 10 were
obtained. In the figure, the graph indicated as "untreated" means
cells which have not undergone the shRNA treatment, the graph
indicated as "control" means cells which have been treated only
with a detection antibody <anti-human IgG-Alexa647
(Invitrogen)>, and the graphs indicated as shITGA6 and shITGB4
mean samples in which the expression of each gene has been
inhibited by shRNA.
(1) Results of Gene Expression Inhibition by ShITGA6 (UCSD/AML1:
ShITGA6)
[0126] As a result of the inhibition of the expression of ITGA6,
the reactivity of the anti-ITGB4 positive control was not changed
(D. BL-ITGB4), whereas the reactivity of the anti-ITGA6BA
antibodies of the present invention almost disappeared (A.
PPMXITG-001, B. PPMXITG-004, and C. PPMXITG-005).
(2) Results of Gene Expression Inhibition by ShITgb4 (UCSD/AML1:
ShITgb4)
[0127] As a result of the inhibition of the expression of ITGB4,
the reactivity of the anti-ITGA6 positive control was not changed
(D. BLITGA6), whereas the reactivity of the anti-ITGA6BA antibodies
of the present invention almost disappeared (A. PPMXITG-001, B.
PPMXITG-004, and C. PPMXITG-005).
(3) Conclusion Regarding Expression Inhibition Tests, and
Conclusion and Consideration Regarding Complex-Specific
Recognition
[0128] In the present example, the expression of ITGA6 and B4 in
AML1 cells as analysis target cells was inhibited at an efficiency
of approximately 50% to 60%. With regard to cells into which the
expression inhibition has been successfully introduced, the cells
are gated with GFP, so that only the expression-inhibited cells can
be analyzed. Taking into consideration these matters, as described
in (1) and (2) above, it was found that the antibody of the present
invention lost its reactivity in both tests of examining the
inhibition of the expression of ITGA6 and ITGB4. Also considering
the results from Examples 2, 4 and 5, it was demonstrated that the
antibody of the present invention recognizes only an ITGA6B4
complex, and does not recognize ITGA6 alone or ITGB4 alone.
Example 12
Verification of Therapeutic Effects of Antibody in Vein Transplant
Models (Infiltration-Inhibiting Ability)
[0129] Using immunodeficient mice (NOG mice), vein transplant
models of the refractory leukemia cell lines UCSD/AML1 and HNT34
were produced. The model mice were administered with a drug (AraC),
an antibody, or the combined use thereof, and the effect of
inhibiting bone marrow infiltration by such administration was then
examined. After the leukemia cell line had been injected into mouse
vein, the mice were administered with PPMXITG-005. Thereafter, the
organ was removed from each experimental body, and the infiltration
level of the human cells was then analyzed. Specifically, the
removed organ was ground with a cell strainer (BD 352340), the
separated cells were then obtained, and erythrocytes were then
hemolyzed with a hemolytic agent (BD 555899). The cells
(1.times.10.sup.6) were suspended in 100 .mu.L of MACS Buffer
(PBS(-) containing 2 mM EDTA and 5% BSA), and were then reacted
with a human CD45 antibody (PE-label, BioLegend 304008), in order
to identify the human cells. Thereafter, the resultant was washed
with MACS Buffer three times, and was then evaluated with a flow
cytometer (BD FACS Calibur).
[0130] Using infiltration of the leukemia cells into the bone
marrow as an indicator, the medicinal effects obtained by the
combined use of the developed antibody and the drug, or by the
single use thereof, were evaluated. As a result, it was found that
infiltration of the leukemia cells into the bone marrow was
significantly inhibited by the combined use of the antibody and the
drug or by the single use thereof. From these results, it is
considered that inhibition of the adhesion of the leukemia cells to
the bone marrow niche by the present antibody is useful for
inhibiting the achievement of cell adhesion-dependent resistance to
chemotherapy (FIG. 11).
Example 13
Effect of ITGA6B4 IgG Antibody to Inhibit In Vitro Cell Growth
[0131] ITGA6B4-expressing cells AML1 and SW480 were each prepared
to a density of 1000 cells/well with a culture solution, and the
obtained solution was then dispensed in an amount of 100 .mu.L/well
each, into a 96-well flat-bottom plate (NUNC 167008). The cells
were cultured at 37.degree. C. in the presence of 5% CO2 and 95%
air for 24 hours. A 10 .mu.g/mL ITGA6B4 antibody was produced, and
100 .mu.L of the antibody was then added to the plate during the
culture. The obtained mixture was further cultured at 37.degree. C.
in the presence of 5% CO2 and 95% air for 96 hours. After
completion of the culture, 10 .mu.L of Cell counting kit-8 (DOJIN
CK04) was added to the culture, and while observing color
development, the mixture was reacted at 37.degree. C. in the
presence of 5% CO2 and 95% air for several hours. Thereafter, using
a plate reader, the absorbance at 450 nm was measured.
Growth rate=Absorbance of antibody-added well/absorbance of
antibody-not-added well.times.100%
[0132] From these analysis results, the effect of each ITGA6B4
antibody to inhibit the cell growth was not observed (FIG. 12).
These results demonstrate that the antibody alone does not have the
medicinal effect of inhibiting the growth of cells, and thus, it is
suggested that the antibody effectively acts regarding the
reduction in side effects of the present invention.
Example 14
Test Regarding Combined Use of Antibody and Anticancer Agent Using
Vein Transplant Models
[0133] In order to confine the effects obtained by the combined use
of the antibody and the drug in vivo, an experiment was carried out
using vein transplant models. Two types of leukemia cell lines
(UCSD/AML1 and EINT34) were each transplanted into severe
immunodeficient mice (NOG mice: IL2R.gamma.KO mice) through the
vein. Regarding experimental conditions, four conditions, namely,
an untreated control, a single use of the anticancer agent, a
single use of the antibody, and the combined use of the antibody
and the anticancer agent, were determined and studied. Regarding
administration conditions, the antibody was administered at a dose
of 3 mg/kg once a week, the anticancer agent was administered at a
dose of 150 mg/kg once a week, and the medicinal effects were
examined based on the survival rate of n=5 for each condition. As a
result, the control group and the single antibody administration
group had the same survival rate, and the survival rate was
increased in the single agent administration group. Moreover, in
the combined use group in which the anticancer agent was used in
combination with the antibody, significant extension of the
survival rate was observed, in compared to the single use of the
anticancer agent (FIG. 13).
[0134] Together with the results of Example 12 (FIG. 11), in which
infiltration of leukemia cells into the bone marrow was analyzed
after intravenous administration of the leukemia cells to mice, it
has been considered that the antibody of the present invention has
the following medicinal effects: (1) the combined use of the
antibody and an anticancer agent inhibits infiltration of leukemia
cells that have been administered through the vein into the bone
marrow; and (2) as a result, the increase of the survival rate is
observed in mouse models. Accordingly, the usefulness of the
antibody of the present invention has been demonstrated.
Sequence CWU 1
1
3115PRTHuman 1Asp Tyr Tyr Met Ser 1 5 217PRTHuman 2Tyr Ile Ser Ser
Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
310PRTHuman 3Trp Asp Thr Ala Met Ala Thr Phe Asp Tyr 1 5 10
413PRTHuman 4Ser Gly Ser Ser Ser Asn Ile Gly Asn Ser Tyr Val Ser 1
5 10 57PRTHuman 5Asp Asn Asn Lys Arg Pro Ser 1 5 611PRTHuman 6Gly
Thr Trp Asp Ser Ser Leu Ser Ala Tyr Val 1 5 10 75PRTHuman 7Ser Tyr
Ala Ile Ser 1 5 817PRTHuman 8Gly Ile Ile Pro Thr Phe Gly Thr Ala
Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 913PRTHuman 9Ser Thr Gly
Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 1011PRTHuman 10Arg
Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 117PRTHuman 11Ala
Ala Ser Ser Leu Gln Ser 1 5 129PRTHuman 12Gln Gln Ser Tyr Ser Thr
Pro Phe Thr 1 5 135PRTHuman 13Ser His Tyr Ile Asn 1 5 1416PRTHuman
14Val Ile Tyr Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Arg Gly 1
5 10 15 156PRTHuman 15Gly Gly Gly Leu Asp Val 1 5 1614PRTHuman
16Thr Leu Arg Ser Asp Val Asn Val Gly Thr Tyr Lys Ile Tyr 1 5 10
1711PRTHuman 17Tyr Asn Ser Asp Ser Asp Lys Gln Gln Gly Ser 1 5 10
1811PRTHuman 18Tyr Cys Met Ile Arg His Asn Ser Ala Trp Val 1 5 10
195PRTHuman 19Gly Tyr Tyr Met His 1 5 2017PRTHuman 20Ile Ile Asn
Pro Ser Gly Gly Thr Thr Arg Leu Ala Gln Lys Phe Gln 1 5 10 15 Gly
2112PRTHuman 21Glu Ala His Ser Ser Gly Ser Tyr Phe Phe Asp Tyr 1 5
10 2211PRTHuman 22Arg Ala Ser Gln Ser Ile Ser Thr Trp Leu Ala 1 5
10 237PRTHuman 23Gln Ala Ser Thr Leu Thr Ser 1 5 2410PRTHuman 24Gln
Glu Tyr Asn Ser Tyr Ser Pro Trp Ala 1 5 10 255PRTHuman 25Asp Tyr
Ala Met His 1 5 2617PRTHuman 26Gly Ile Ser Trp Asn Ser Gly Ser Ile
Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2713PRTHuman 27Ala Leu
Pro Ser Gly Gly Trp Ala Asp Ala Phe Asp Ile 1 5 10 2813PRTHuman
28Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Tyr Val Gln 1 5 10
297PRTHuman 29Glu Asp Asp Gln Arg Pro Ser 1 5 309PRTHuman 30Gln Ser
Phe His Asp Thr Lys Gln Met 1 5 3115PRTArtificial
SequenceDescription of Artificial Sequence linker 31Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
* * * * *
References